U.S. patent application number 12/994921 was filed with the patent office on 2011-08-04 for use of zinc chelators to inhibit biofilm formation.
This patent application is currently assigned to UNIVERSITY OF CINCINNATI. Invention is credited to Cristin C. Brescia, Deborah Gail Conrady, Andrew B. Herr, Stefanie L. Ward.
Application Number | 20110189260 12/994921 |
Document ID | / |
Family ID | 41434389 |
Filed Date | 2011-08-04 |
United States Patent
Application |
20110189260 |
Kind Code |
A1 |
Herr; Andrew B. ; et
al. |
August 4, 2011 |
USE OF ZINC CHELATORS TO INHIBIT BIOFILM FORMATION
Abstract
A method is provided for inhibiting formation of a biofilm of
bacteria, the method including contacting the bacteria with an
effective amount of at least one zinc chelator, wherein the
bacteria contain at least one zinc adhesion module, whereby
formation of the biofilm is inhibited. A method for inhibiting
biofilm formation on a device and a topical pharmaceutical
composition for the inhibition of biofilm formation are also
provided.
Inventors: |
Herr; Andrew B.;
(Cincinnati, OH) ; Conrady; Deborah Gail;
(Cincinnati, OH) ; Brescia; Cristin C.; (Canton,
OH) ; Ward; Stefanie L.; (Cincinnati, OH) |
Assignee: |
UNIVERSITY OF CINCINNATI
Cincinnati
OH
|
Family ID: |
41434389 |
Appl. No.: |
12/994921 |
Filed: |
May 29, 2009 |
PCT Filed: |
May 29, 2009 |
PCT NO: |
PCT/US09/45623 |
371 Date: |
April 4, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61057267 |
May 30, 2008 |
|
|
|
Current U.S.
Class: |
424/447 ; 424/54;
514/292; 514/566; 514/638 |
Current CPC
Class: |
C11D 3/38 20130101; C11D
3/48 20130101; A01N 37/44 20130101; C11D 3/33 20130101; A01N 43/90
20130101; A61P 31/02 20180101; A01N 43/40 20130101; A01N 47/14
20130101; A01N 37/44 20130101; A61P 31/04 20180101; A01N 37/44
20130101; A01N 2300/00 20130101; A01N 25/34 20130101 |
Class at
Publication: |
424/447 ;
514/566; 514/292; 514/638; 424/54 |
International
Class: |
A01N 37/12 20060101
A01N037/12; A01N 43/42 20060101 A01N043/42; A01N 33/02 20060101
A01N033/02; A61K 9/00 20060101 A61K009/00; A61K 31/195 20060101
A61K031/195; A61K 31/4375 20060101 A61K031/4375; A61K 31/13
20060101 A61K031/13; A01P 1/00 20060101 A01P001/00; A61P 31/04
20060101 A61P031/04 |
Claims
1. A method for inhibiting formation of a biofilm comprising
bacteria, the method comprising contacting the bacteria with an
effective amount of at least one zinc chelator, wherein the
bacteria comprise at least one zinc adhesion module, whereby
formation of the biofilm is inhibited.
2. The method of claim 1 wherein the bacteria are selected from the
group consisting of gram-positive bacteria.
3. The method of claim 1 wherein the bacteria are selected from the
group consisting of Acidothermus cellulyticus, Actinomyces
odontolyticus, Alkaliphilus metalliredigens, Alkaliphilus
oremlandii, Arthrobacter aurescens, Bacillus amyloliquefaciens,
Bacillus clausii, Bacillus halodurans, Bacillus licheniformis,
Bacillus pumilus, Bacillus subtilis, Bifidobacterium adolescentis,
Bifidiobacterium longum, Caldicellulosiruptor saccharolyticus,
Carboxydothermus hydrogenoformans, Clostridium acetobutylicum,
Clostridium beijerinckii, Clostridium botulinum, Clostridium
cellulolyticum, Clostridium difficile, Clostridium kluyveri,
Clostridium leptum, Clostridium novyi, Clostridium perfringens,
Clostridium tetani, Clostridium thermocellum, Corynebacterium
diphtheriae, Corynebacterium efficiens, Corynebacterium glutamicum,
Corynebacterium jeikeium, Corynebacterium urealyticum,
Desulfitobacterium hafniense, Desulfotomaculum reducens,
Eubacterium ventriosum, Exiguobacterium sibiricum, Fingoldia magna,
Geobacillus kaustophilus, Geobacillus thermodenitrificans,
Janibacter sp., Kineococcus radiotolerans, Lactobacillus fermentum,
Listeria monocytogenes, Listeria innocua, Listeria welshimeri,
Moorella thermoacetica, Mycobacterium avium, Mycobacterium bovis,
Mycobacterium gilvum, Mycobacterium leprae, Mycobacterium
paratuberculosis, Mycobacterium smegmatis, Mycobacterium
tuberculosis, Mycobacterium ulcerans, Mycobacterium vanbaalenii,
Nocardioides sp., Nocardia farcinica, Oceanobacillus iheyensis,
Pelotomaculum thermopropionicum, Rhodococcus sp., Saccharopolyspora
erythraea, coagulase-negative Staphylococcus species,
Staphylococcus aureus, methicillin resistant Staphylococcus aureus
(MRSA), Staphylococcus epidermidis, methicillin resistant
Staphylococcus epidermidis (MRSE), Streptococcus agalactiae,
Streptococcus gordonii, Streptococcus mitis, Streptococcus oralis,
Streptococcus pneumoniae, Streptococcus sanguinis, Streptococcus
suis, Streptomyces avermitilis, Streptomyces coelicolor,
Thermoanaerobacter ethanolicus, Thermoanaerobacter tengcongensis,
and combinations thereof.
4. The method of claim 3 wherein the bacteria are selected from the
group consisting of Corynebacterium urealyticum, Fingoldia magna,
Staphylococcus aureus, methicillin resistant Staphylococcus aureus
(MRSA), Staphylococcus epidermidis, methicillin resistant
Staphylococcus epidermidis (MRSE), Streptococcus gordonii,
Streptococcus pneumoniae, Streptococcus sanguinis, Streptococcus
suis, and combinations thereof.
5. The method of claim 4 wherein the bacteria are selected from the
group consisting of Staphylococcus aureus, methicillin resistant
Staphylococcus aureus (MRSA), Staphylococcus epidermidis,
methicillin resistant Staphylococcus epidermidis (MRSE), and
combinations thereof.
6. The method of claim 1 wherein the zinc chelator is selected from
the group consisting of EDTA, DTPA, TPEN, 1,10-phenanthroline,
clioquinol, diethyldithiocarbamate (DEDTC), DMPS, EDPA, DMHP, DEHP,
EM, TFLZn, dithiozone, TSQ, carnosine, deferasirox, CyDTA, DHEG,
DTPA-OH, EDDA, EDDP, EDDPO, EDTA-OH, EDTPO, EGTA, HBED, HDTA, HIDA,
IDA, Methyl-EDTA, NTA, NTP, NTPO, O-Bistren, TTHA, DMSA,
deferoxamine, dimercaprol, zinc citrate, combination of bismuth and
citrate, penicilamine, succimer, Etidronate, EDDHA, CDTA, HEDTA,
HEIDA, calprotectin, zinc fingers, lactoferrin, ovotransferrin,
conalbumin, and combinations thereof.
7. The method of claim 6 wherein the zinc chelator is selected from
the group consisting of DTPA, TPEN, 1,10 phenanthroline, EDTA,
DEDTC, EDDA and combinations thereof.
8. The method of claim 7 wherein the zinc chelator is DTPA.
9. A method for inhibiting formation of a biofilm on a device,
wherein the biofilm comprises bacteria comprising at least one zinc
adhesion module, the method comprising contacting the device with a
solution comprising an effective amount of at least one zinc
chelator, whereby formation of a biofilm on the device is
inhibited.
10. The method of claim 9 wherein the device comprises an
implantable medical device.
11. The method of claim 10 wherein the implantable medical device
is selected from the group consisting of pacemakers, heart valves,
replacement joints, catheters, catheter access ports, dialysis
tubing, gastric bands, shunts, screw plates, artificial spinal disc
replacements, internal implantable defibrillators, cardiac
resynchronization therapy devices, implantable cardiac monitors,
mitral valve ring repair devices, left ventricular assist devices
(LVADs), artificial hearts, implantable infusion pumps, implantable
insulin pumps, stents, implantable neurostimulators, maxillofacial
implants, and dental implants.
12. The method of claim 9 wherein the bacteria are selected from
the group consisting of Acidothermus cellulyticus, Actinomyces
odontolyticus, Alkaliphilus metalliredigens, Alkaliphilus
oremlandii, Arthrobacter aurescens, Bacillus amyloliquefaciens,
Bacillus clausii, Bacillus halodurans, Bacillus licheniformis,
Bacillus pumilus, Bacillus subtilis, Bifidobacterium adolescentis,
Bifidiobacterium longum, Caldicellulosiruptor saccharolyticus,
Carboxydothermus hydrogenoformans, Clostridium acetobutylicum,
Clostridium beijerinckii, Clostridium botulinum, Clostridium
cellulolyticum, Clostridium difficile, Clostridium kluyveri,
Clostridium leptum, Clostridium novyi, Clostridium perfringens,
Clostridium tetani, Clostridium thermocellum, Corynebacterium
diphtheriae, Corynebacterium efficiens, Corynebacterium glutamicum,
Corynebacterium jeikeium, Corynebacterium urealyticum,
Desulfitobacterium hafniense, Desulfotomaculum reducens,
Eubacterium ventriosum, Exiguobacterium sibiricum, Fingoldia magna,
Geobacillus kaustophilus, Geobacillus thermodenitrificans,
Janibacter sp., Kineococcus radiotolerans, Lactobacillus fermentum,
Listeria monocytogenes, Listeria innocua, Listeria welshimeri,
Moorella thermoacetica, Mycobacterium avium, Mycobacterium bovis,
Mycobacterium gilvum, Mycobacterium leprae, Mycobacterium
paratuberculosis, Mycobacterium smegmatis, Mycobacterium
tuberculosis, Mycobacterium ulcerans, Mycobacterium vanbaalenii,
Nocardioides sp., Nocardia farcinica, Oceanobacillus iheyensis,
Pelotomaculum thermopropionicum, Rhodococcus sp., Saccharopolyspora
erythraea, coagulase-negative Staphylococcus species,
Staphylococcus aureus, methicillin resistant Staphylococcus aureus
(MRSA), Staphylococcus epidermidis, methicillin resistant
Staphylococcus epidermidis (MRSE), Streptococcus agalactiae,
Streptococcus gordonii, Streptococcus mitis, Streptococcus oralis,
Streptococcus pneumoniae, Streptococcus sanguinis, Streptococcus
suis, Streptomyces avermitilis, Streptomyces coelicolor,
Thermoanaerobacter ethanolicus, Thermoanaerobacter tengcongensis,
and combinations thereof.
13. The method of claim 12 wherein the bacteria are selected from
the group consisting of Corynebacterium urealyticum, Fingoldia
magna, Staphylococcus aureus, methicillin resistant Staphylococcus
aureus (MRSA), Staphylococcus epidermidis, methicillin resistant
Staphylococcus epidermidis (MRSE), Streptococcus gordonii,
Streptococcus pneumoniae, Streptococcus sanguinis, Streptococcus
suis, and combinations thereof.
14. The method of claim 13 wherein the bacteria are selected from
the group consisting of Staphylococcus aureus, methicillin
resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis,
methicillin resistant Staphylococcus epidermidis (MRSE), and
combinations thereof.
15. The method of claim 9 wherein the zinc chelator is selected
from the group consisting of EDTA, DTPA, TPEN, 1,10-phenanthroline,
clioquinol, diethyldithiocarbamate (DEDTC), DMPS, EDPA, DMHP, DEHP,
EM, TFLZn, dithiozone, TSQ, carnosine, deferasirox, CyDTA, DHEG,
DTPA-OH, EDDA, EDDP, EDDPO, EDTA-OH, EDTPO, EGTA, HBED, HDTA, HIDA,
IDA, Methyl-EDTA, NTA, NTP, NTPO, O-Bistren, TTHA, DMSA,
deferoxamine, dimercaprol, zinc citrate, combination of bismuth and
citrate, penicilamine, succimer, Etidronate, EDDHA, CDTA, HEDTA,
HEIDA, calprotectin, zinc fingers, lactoferrin, ovotransferrin,
conalbumin, and combinations thereof.
16. The method of claim 15 wherein the zinc chelator is selected
from the group consisting of DTPA, TPEN, 1,10 phenanthroline, EDTA,
DEDTC, EDDA and combinations thereof.
17. The method of claim 9 wherein the contacting comprises bathing
or coating the device.
18. The method of claim 17 wherein the solution is a gel or polymer
coating.
19. A topical pharmaceutical composition for inhibiting formation
of a biofilm in a mammal, wherein the biofilm comprises bacteria
comprising at least one zinc adhesion module, the pharmaceutical
composition comprising a therapeutically effective amount of at
least one zinc chelator and at least one pharmaceutically
acceptable carrier.
20. The pharmaceutical composition of claim 19, wherein the
composition is a spray, gel, cream, solution, lotion, or
ointment.
21. The pharmaceutical composition of claim 19, wherein the
bacteria are selected from the group consisting of Acidothermus
cellulyticus, Actinomyces odontolyticus, Alkaliphilus
metalliredigens, Alkaliphilus oremlandii, Arthrobacter aurescens,
Bacillus amyloliquefaciens, Bacillus clausii, Bacillus halodurans,
Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis,
Bifidobacterium adolescentis, Bifidiobacterium longum,
Caldicellulosiruptor saccharolyticus, Carboxydothermus
hydrogenoformans, Clostridium acetobutylicum, Clostridium
beijerinckii, Clostridium botulinum, Clostridium cellulolyticum,
Clostridium difficile, Clostridium kluyveri, Clostridium leptum,
Clostridium novyi, Clostridium perfringens, Clostridium tetani,
Clostridium thermocellum, Corynebacterium diphtheriae,
Corynebacterium efficiens, Corynebacterium glutamicum,
Corynebacterium jeikeium, Corynebacterium urealyticum,
Desulfitobacterium hafniense, Desulfotomaculum reducens,
Eubacterium ventriosum, Exiguobacterium sibiricum, Fingoldia magna,
Geobacillus kaustophilus, Geobacillus thermodenitrificans,
Janibacter sp., Kineococcus radiotolerans, Lactobacillus fermentum,
Listeria monocytogenes, Listeria innocua, Listeria welshimeri,
Moorella thermoacetica, Mycobacterium avium, Mycobacterium bovis,
Mycobacterium gilvum, Mycobacterium leprae, Mycobacterium
paratuberculosis, Mycobacterium smegmatis, Mycobacterium
tuberculosis, Mycobacterium ulcerans, Mycobacterium vanbaalenii,
Nocardioides sp., Nocardia farcinica, Oceanobacillus iheyensis,
Pelotomaculum thermopropionicum, Rhodococcus sp., Saccharopolyspora
erythraea, coagulase-negative Staphylococcus species,
Staphylococcus aureus, methicillin resistant Staphylococcus aureus
(MRSA), Staphylococcus epidermidis, methicillin resistant
Staphylococcus epidermidis (MRSE), Streptococcus agalactiae,
Streptococcus gordonii, Streptococcus mitis, Streptococcus oralis,
Streptococcus pneumoniae, Streptococcus sanguinis, Streptococcus
suis, Streptomyces avermitilis, Streptomyces coelicolor,
Thermoanaerobacter ethanolicus, Thermoanaerobacter tengcongensis,
and combinations thereof.
22. The pharmaceutical composition of claim 21, wherein the
bacteria are selected from the group consisting of Corynebacterium
urealyticum, Fingoldia magna, Staphylococcus aureus, methicillin
resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis,
methicillin resistant Staphylococcus epidermidis (MRSE),
Streptococcus gordonii, Streptococcus pneumoniae, Streptococcus
sanguinis, Streptococcus suis, and combinations thereof.
23. The pharmaceutical composition of claim 22, wherein the
bacteria are selected from the group consisting of Staphylococcus
aureus, methicillin resistant Staphylococcus aureus (MRSA),
Staphylococcus epidermidis, methicillin resistant Staphylococcus
epidermidis (MRSE), and combinations thereof.
24. The pharmaceutical composition of claim 19, wherein the zinc
chelator is selected from the group consisting of EDTA, DTPA, TPEN,
1,10-phenanthroline, clioquinol, diethyldithiocarbamate (DEDTC),
DMPS, EDPA, DMHP, DEHP, EM, TFLZn, dithiozone, TSQ, carnosine,
deferasirox, CyDTA, DHEG, DTPA-OH, EDDA, EDDP, EDDPO, EDTA-OH,
EDTPO, EGTA, HBED, HDTA, HIDA, IDA, Methyl-EDTA, NTA, NTP, NTPO,
O-Bistren, TTHA, DMSA, deferoxamine, dimercaprol, zinc citrate,
combination of bismuth and citrate, penicilamine, succimer,
Etidronate, EDDHA, CDTA, HEDTA, HEIDA, calprotectin, zinc fingers,
lactoferrin, ovotransferrin, conalbumin, and combinations
thereof.
25. The pharmaceutical composition of claim 24, wherein the zinc
chelator is selected from the group consisting of DTPA, TPEN, 1,10
phenanthroline, EDTA, DEDTC, EDDA and combinations thereof.
26. The pharmaceutical composition of claim 19, further comprising
a therapeutically effective amount of at least one antimicrobial
agent.
27. The pharmaceutical composition of claim 26, wherein the
antimicrobial agent is an antibiotic.
28. A surgical rinse for inhibiting formation of a biofilm
comprising bacteria, wherein the bacteria comprise at least one
zinc adhesion module, the surgical rinse comprising an effective
amount of at least one zinc chelator.
29. The surgical rinse of claim 28, wherein the bacteria are
selected from the group consisting of Acidothermus cellulyticus,
Actinomyces odontolyticus, Alkaliphilus metalliredigens,
Alkaliphilus oremlandii, Arthrobacter aurescens, Bacillus
amyloliquefaciens, Bacillus clausii, Bacillus halodurans, Bacillus
licheniformis, Bacillus pumilus, Bacillus subtilis, Bifidobacterium
adolescentis, Bifidiobacterium longum, Caldicellulosiruptor
saccharolyticus, Carboxydothermus hydrogenoformans, Clostridium
acetobutylicum, Clostridium beijerinckii, Clostridium botulinum,
Clostridium cellulolyticum, Clostridium difficile, Clostridium
kluyveri, Clostridium leptum, Clostridium novyi, Clostridium
perfringens, Clostridium tetani, Clostridium thermocellum,
Corynebacterium diphtheriae, Corynebacterium efficiens,
Corynebacterium glutamicum, Corynebacterium jeikeium,
Corynebacterium urealyticum, Desulfitobacterium hafniense,
Desulfotomaculum reducens, Eubacterium ventriosum, Exiguobacterium
sibiricum, Fingoldia magna, Geobacillus kaustophilus, Geobacillus
thermodenitrificans, Janibacter sp., Kineococcus radiotolerans,
Lactobacillus fermentum, Listeria monocytogenes, Listeria innocua,
Listeria welshimeri, Moorella thermoacetica, Mycobacterium avium,
Mycobacterium bovis, Mycobacterium gilvum, Mycobacterium leprae,
Mycobacterium paratuberculosis, Mycobacterium smegmatis,
Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycobacterium
vanbaalenii, Nocardioides sp., Nocardia farcinica, Oceanobacillus
iheyensis, Pelotomaculum thermopropionicum, Rhodococcus sp.,
Saccharopolyspora erythraea, coagulase-negative Staphylococcus
species, Staphylococcus aureus, methicillin resistant
Staphylococcus aureus (MRSA), Staphylococcus epidermidis,
methicillin resistant Staphylococcus epidermidis (MRSE),
Streptococcus agalactiae, Streptococcus gordonii, Streptococcus
mitis, Streptococcus oralis, Streptococcus pneumoniae,
Streptococcus sanguinis, Streptococcus suis, Streptomyces
avermitilis, Streptomyces coelicolor, Thermoanaerobacter
ethanolicus, Thermoanaerobacter tengcongensis, and combinations
thereof.
30. The surgical rinse of claim 29, wherein the bacteria are
selected from the group consisting of Corynebacterium urealyticum,
Fingoldia magna, Staphylococcus aureus, methicillin resistant
Staphylococcus aureus (MRSA), Staphylococcus epidermidis,
methicillin resistant Staphylococcus epidermidis (MRSE),
Streptococcus gordonii, Streptococcus pneumoniae, Streptococcus
sanguinis, Streptococcus suis, and combinations thereof.
31. The surgical rinse of claim 30, wherein the bacteria are
selected from the group consisting of Staphylococcus aureus,
methicillin resistant Staphylococcus aureus (MRSA), Staphylococcus
epidermidis, methicillin resistant Staphylococcus epidermidis
(MRSE), and combinations thereof.
32. The surgical rinse of claim 28, wherein the zinc chelator is
selected from the group consisting of EDTA, DTPA, TPEN,
1,10-phenanthroline, clioquinol, diethyldithiocarbamate (DEDTC),
DMPS, EDPA, DMHP, DEHP, EM, TFLZn, dithiozone, TSQ, carnosine,
deferasirox, CyDTA, DHEG, DTPA-OH, EDDA, EDDP, EDDPO, EDTA-OH,
EDTPO, EGTA, HBED, HDTA, HIDA, IDA, Methyl-EDTA, NTA, NTP, NTPO,
O-Bistren, TTHA, DMSA, deferoxamine, dimercaprol, zinc citrate,
combination of bismuth and citrate, penicilamine, succimer,
Etidronate, EDDHA, CDTA, HEDTA, HEIDA, calprotectin, zinc fingers,
lactoferrin, ovotransferrin, conalbumin and combinations
thereof.
33. The surgical rinse of claim 32, wherein the zinc chelator is
selected from the group consisting of DTPA, TPEN, 1,10
phenanthroline, EDTA, DEDTC, EDDA and combinations thereof.
34. A method for inhibiting formation of a biofilm comprising
bacteria, the method comprising contacting the bacteria with an
effective amount of at least one zinc chelator, wherein the
bacteria are selected from the group consisting of Staphylococcus
aureus, methicillin resistant Staphylococcus aureus (MRSA),
Staphylococcus epidermidis, methicillin resistant Staphylococcus
epidermidis (MRSE), Streptococcus sanguinis, and Streptococcus
suis, whereby formation of the biofilm is inhibited.
35. The method of claim 34 wherein the zinc chelator is selected
from the group consisting of DTPA, TPEN, 1,10 phenanthroline, EDTA,
DEDTC, EDDA and combinations thereof.
36. A method for inhibiting the formation of a biofilm comprising
bacteria, wherein the bacteria comprise at least one zinc adhesion
module, the method comprising contacting the bacteria with a
composition comprising at least one soluble zinc adhesion module,
whereby formation of the biofilm is inhibited.
37. The method of claim 36, wherein the bacteria are selected from
the group consisting of Acidothermus cellulyticus, Actinomyces
odontolyticus, Alkaliphilus metalliredigens, Alkaliphilus
oremlandii, Arthrobacter aurescens, Bacillus amyloliquefaciens,
Bacillus clausii, Bacillus halodurans, Bacillus licheniformis,
Bacillus pumilus, Bacillus subtilis, Bifidobacterium adolescentis,
Bifidiobacterium longum, Caldicellulosiruptor saccharolyticus,
Carboxydothermus hydrogenoformans, Clostridium acetobutylicum,
Clostridium beijerinckii, Clostridium botulinum, Clostridium
cellulolyticum, Clostridium difficile, Clostridium kluyveri,
Clostridium leptum, Clostridium novyi, Clostridium perfringens,
Clostridium tetani, Clostridium thermocellum, Corynebacterium
diphtheriae, Corynebacterium efficiens, Corynebacterium glutamicum,
Corynebacterium jeikeium, Corynebacterium urealyticum,
Desulfitobacterium hafniense, Desulfotomaculum reducens,
Eubacterium ventriosum, Exiguobacterium sibiricum, Fingoldia magna,
Geobacillus kaustophilus, Geobacillus thermodenitrificans,
Janibacter sp., Kineococcus radiotolerans, Lactobacillus fermentum,
Listeria monocytogenes, Listeria innocua, Listeria welshimeri,
Moorella thermoacetica, Mycobacterium avium, Mycobacterium bovis,
Mycobacterium gilvum, Mycobacterium leprae, Mycobacterium
paratuberculosis, Mycobacterium smegmatis, Mycobacterium
tuberculosis, Mycobacterium ulcerans, Mycobacterium vanbaalenii,
Nocardioides sp., Nocardia farcinica, Oceanobacillus iheyensis,
Pelotomaculum thermopropionicum, Rhodococcus sp., Saccharopolyspora
erythraea, coagulase-negative Staphylococcus species,
Staphylococcus aureus, methicillin resistant Staphylococcus aureus
(MRSA), Staphylococcus epidermidis, methicillin resistant
Staphylococcus epidermidis (MRSE), Streptococcus agalactiae,
Streptococcus gordonii, Streptococcus mitis, Streptococcus oralis,
Streptococcus pneumoniae, Streptococcus sanguinis, Streptococcus
suis, Streptomyces avermitilis, Streptomyces coelicolor,
Thermoanaerobacter ethanolicus, Thermoanaerobacter tengcongensis,
and combinations thereof.
38. The method of claim 37, wherein the bacteria are selected from
the group consisting of Corynebacterium urealyticum, Fingoldia
magna, Staphylococcus aureus, methicillin resistant Staphylococcus
aureus (MRSA), Staphylococcus epidermidis, methicillin resistant
Staphylococcus epidermidis (MRSE), Streptococcus gordonii,
Streptococcus pneumoniae, Streptococcus sanguinis, Streptococcus
suis, and combinations thereof.
39. The method of claim 38, wherein the bacteria are selected from
the group consisting of Staphylococcus aureus, methicillin
resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis,
methicillin resistant Staphylococcus epidermidis (MRSE), and
combinations thereof.
40. The method of claim 37 wherein the composition is a spray,
solution, gel, cream, ointment, surgical rinse, or dental
rinse.
41. A bandage impregnated with a safe and effective amount of at
least one zinc chelator, wherein the bandage inhibits the formation
of a biofilm on the skin, wherein the biofilm comprises bacteria
comprising at least one zinc adhesion module.
42. A personal cleansing composition comprising an effective amount
of at least one zinc chelator, wherein the personal cleansing
composition inhibits formation of a biofilm on the skin, wherein
the biofilm comprises bacteria comprising at least one zinc
adhesion module.
43. The personal cleansing composition of claim 42, wherein the
composition is a surgical scrub, shower gel, body wash, or
soap.
44. A hard surface cleaning composition comprising an effective
amount of at least one zinc chelator, wherein the composition
inhibits formation of a biofilm on a hard surface, wherein the
biofilm comprises bacteria comprising at least one zinc adhesion
module.
45. A dental rinse for inhibiting formation of a biofilm wherein
the biofilm comprises bacteria comprising at least one zinc
adhesion module, the dental rinse comprising an effective amount of
at least one zinc chelator.
Description
[0001] This application claims priority under 35 U.S.C. .sctn.119
to U.S. Provisional Application Ser. No. 61/057,267, filed May 30,
2008, which application is incorporated by reference in its
entirety.
[0002] The present invention relates to the fields of microbiology
and biochemistry, and more specifically to methods for inhibiting
the formation of a bacterial biofilm, wherein the targeted bacteria
comprise at least one zinc adhesion module, the method comprising
contacting the bacteria with an effective amount of at least one
zinc chelator, whereby biofilm formation is inhibited.
[0003] It has recently been estimated that hospital-acquired
(nosocomial) infections are the fourth-leading cause of death in
the United States, affecting 2 million patients per year and
causing over 100,000 annual deaths, with a total annual cost of
over $30 billion. Staphylococcal species such as S. epidermidis and
S. aureus are responsible for the majority of nosocomial
infections; treatment of these infections is often made much more
challenging by the tendency of staphylococci to form biofilms.
[0004] Biofilms are bacterial communities that adhere to biological
or abiotic substrata, differentiate into micro- and macrocolonies,
and produce an extracellular matrix typically comprised of
polysaccharides and proteins. Bacteria in biofilms are resistant to
antibiotics and host immune responses and are extremely difficult
to eradicate. For example, device-related infections due to
staphylococcal biofilms often require surgical removal of the
implanted device, debridement of the surrounding tissue, and
prolonged antibiotic treatment.
[0005] Staphylococcal infections are a substantial re-emerging
threat to human health. Starting in the 1980's a dramatic increase
in hospital-acquired infections has been observed, primarily
associated with S. epidermidis and other coagulase-negative
staphylococci. S. epidermidis alone is responsible for up to
two-thirds of all central nervous system shunt- or catheter-related
infections, as well as up to half of all infections of prosthetic
heart valves or artificial joints. The nosocomial infections caused
by staphylococci are often persistent and recurring, particularly
in the case of infections of indwelling medical devices.
Furthermore, device-related S. epidermidis biofilms can lead to
endocarditis or septicemia. Likewise, S. aureus biofilm-related
infections can become disseminated to other regions, which is a
concern due to the large number of toxins and tissue-degrading
enzymes released by S. aureus.
[0006] Industrially, biofilms can accumulate on a wide variety of
substrates and interfere in a number of industrial and commercial
applications, including pipeline systems, cooling water systems in
power plants, refineries, chemical plants, air conditioning
systems, and the like. If uncontrolled, biofouling caused by
biofilm formation can interfere with operations, lower efficiency
of systems, waste energy and resources, and adversely affect
product quality.
[0007] A need exists to control and inhibit biofilm formation in
medical and industrial applications. One area of current interest
involves the use of metal chelation as a means of inhibiting
biofilms. The present invention relates specifically to the
chelation of zinc metal ions in biofilms comprised of bacteria
having a zinc adhesion module.
[0008] Methods of inhibiting biofilm formation in medical and
industrial settings have previously been developed using metal
chelators, specifically iron chelators. For example, U.S. Pat. No.
6,267,979, issued Jul. 31, 2001, to Raad et al., discloses the use
of metal chelators in combination with antifungal or antibiotic
compositions for the prevention of biofouling in water treatment,
pulp and paper manufacturing and oil field water flooding. U.S.
Pat. No. 7,314,857, issued Jan. 1, 2008, to Madhyastha, discloses
synergistic antimicrobial compositions for inhibiting biofilm
formation using combinations of an iron-sequestering glycoprotein,
a cationic peptide, and an iron chelating agent. U.S. Pat. No.
7,446,089, issued Nov. 4, 2008, to Singh et al., is also directed
to methods of inhibiting biofilm formation by limiting the amount
of iron available to a population of bacteria, such that biofilm
formation can be inhibited. These disclosures generally target
iron, a higher affinity metal ion. Indeed, the specified ranges
disclosed in Madhyastha for metal chelation, such as 12.5 to 200
mg/L, would not be effective for zinc chelation in gram positive
bacteria, as zinc is a lower affinity metal ion requiring
relatively higher concentrations of chelating agents.
[0009] Staphylococcal biofilms are typically enmeshed within an
extracellular polysaccharide matrix synthesized by proteins encoded
by the ica operon; however, ica-negative staphylococcal biofilms
have recently been described that rely on protein-protein
interactions. The S. epidermidis surface protein Aap
(Accumulation-associated protein) has been implicated in both
polysaccharide-based and protein-based S. epidermidis biofilms. Aap
contains an N-terminal A-repeat region with 11 degenerate 16 amino
acid repeats, a putative globular domain (".alpha./.beta."), and a
B-repeat region with a variable number (5 to 17) of nearly
identical 128 amino acid repeats terminating in a conserved "half
repeat" motif (FIG. 1 A). Rohde, H. et al., Polysaccharide
intercellular adhesin or protein factors in biofilm accumulation of
Staphylococcus epidermidis and Staphylococcus aureus isolated from
prosthetic hip and knee joint infections, Biomaterials 28:1711-20
(2007). The repeated sequence element within the B-repeat region
has recently been defined as a G5 domain, found in gram-positive
surface proteins, Zn.sup.2+ metalloproteases, and other bacterial
virulence factors (FIG. 5). The B-repeat region in Aap is followed
by a collagen-like repeat and an LPXTG cell wall anchor sequence. A
similar domain arrangement exists in the S. aureus homolog SasG
(FIG. 1B). Corrigan, R. M. et al., The role of Staphylococcus
aureus surface protein SasG in adherence and biofilm formation,
Microbiology 153:2435-46 (2007).
[0010] Proteolytic processing of Aap or SasG between the
.alpha./.beta. and B-repeat regions induces the formation of
protein-based biofilms in S. epidermidis and S. aureus; both
proteinaceous and polysaccharide-based S. epidermidis biofilms are
inhibited by anti-Aap antisera. Furthermore, addition of a large
soluble Aap fragment containing the G5 and collagen-like regions
inhibited formation of protein-based S. epidermidis biofilms.
Rohde, H. et al., Induction of Staphylococcus epidermidis biofilm
formation via proteolytic processing of the accumulation-associated
protein by staphylococcal and host proteases, Mol. Microbiol.
55:1883-95 (2005). In S. aureus, protein-based biofilm formation
was dependent on the number of G5 domains in SasG; five or more G5
domains were required to support biofilm formation. These studies
suggested that the C-terminal halves of Aap and SasG containing the
G5 domains are involved in bacterial interactions within
staphylococcal biofilms, but the precise molecular mechanism was
unclear.
[0011] Given the serious medical, industrial, and environmental
problems associated with bacterial biofilms, the need persists to
develop targeted approaches to inhibit biofilm formation.
[0012] The present invention discloses a biophysical
characterization of a G5 domain-containing B-repeat region from
Aap, revealing that it is a zinc (Zn.sup.2+)-dependent adhesion
module ("zinc adhesion module") responsible for intercellular
interaction in staphylococcal biofilms. This zinc adhesion module
has been identified in a variety of bacteria, including gram
positive bacteria, and provides a specific target for zinc
chelation and biofilm inhibition in biofilms comprised of bacteria
having a G5 domain.
[0013] Surprisingly, it has been found that zinc chelation inhibits
formation of both S. epidermidis and methicillin-resistant S.
aureus biofilms and supplementation with additional zinc in the
physiological range reverses the effect. This reversible effect
identified in bacteria having a zinc adhesion module, herein
described as a "zinc zipper," underscores the criticality of zinc
in intercellular adhesion, providing a specific target for
chelation and biofilm inhibition. Furthermore, addition of a
soluble Aap fragment containing a single intact zinc adhesion
module inhibits biofilm formation in a dose-dependent manner,
indicating that the G5 domain is indeed a required element for
intercellular adhesion in staphylococcal biofilms.
[0014] Accordingly, the present invention provides a method for
inhibiting formation of a biofilm comprising bacteria, the method
comprising contacting the bacteria with an effective amount of at
least one zinc chelator, wherein the bacteria comprise at least one
zinc adhesion module, whereby formation of the biofilm is
inhibited.
[0015] In a further embodiment, the present invention provides a
method for inhibiting formation of a biofilm on a device, wherein
the biofilm comprises bacteria comprising at least one zinc
adhesion module, the method comprising contacting the device with a
solution comprising an effective amount of at least one zinc
chelator, whereby formation of a biofilm on the device is
inhibited.
[0016] In another embodiment, the present invention provides a
topical pharmaceutical composition for inhibiting formation of a
biofilm on or in a mammal, wherein the biofilm comprises bacteria
comprising at least one zinc adhesion module, the pharmaceutical
composition comprising a therapeutically effective amount of at
least one zinc chelator and at least one pharmaceutically
acceptable carrier.
[0017] In a further embodiment, the present invention provides a
surgical rinse for inhibiting formation of a biofilm comprising
bacteria, wherein the bacteria comprise at least one zinc adhesion
module, the surgical rinse comprising an effective amount of at
least one zinc chelator.
[0018] In still another embodiment, the present invention provides
a method for inhibiting formation of a biofilm comprising bacteria,
the method comprising contacting the bacteria with an effective
amount of at least one zinc chelator, wherein the bacteria are
selected from the group consisting of Staphylococcus aureus,
methicillin resistant Staphylococcus aureus (MRSA), Staphylococcus
epidermidis, methicillin resistant Staphylococcus epidermidis
(MRSE), Streptococcus sanguinis, and Streptococcus suis, whereby
formation of the biofilm is inhibited.
[0019] In another embodiment, the present invention provides a
method for inhibiting the formation of a biofilm comprising
bacteria, wherein the bacteria comprise at least one zinc adhesion
module, the method comprising contacting the bacteria with a
composition comprising at least one soluble zinc adhesion module,
whereby formation of the biofilm is inhibited.
[0020] A further embodiment of the present invention provides a
bandage impregnated with a safe and effective amount of at least
one zinc chelator, wherein the bandage inhibits the formation of a
biofilm on the skin, wherein the biofilm comprises bacteria
comprising at least one zinc adhesion module.
[0021] Still another embodiment of the present invention provides a
personal cleansing composition comprising an effective amount of at
least one zinc chelator, wherein the personal cleansing composition
inhibits formation of a biofilm on the skin, wherein the biofilm
comprises bacteria comprising at least one zinc adhesion
module.
[0022] A further embodiment of the present invention provides a
hard surface cleaning composition comprising an effective amount of
at least one zinc chelator, wherein the composition inhibits
formation of a biofilm on a hard surface, wherein the biofilm
comprises bacteria comprising at least one zinc adhesion
module.
[0023] Still another embodiment of the present invention provides a
dental rinse for inhibiting formation of a biofilm wherein the
biofilm comprises bacteria comprising at least one zinc adhesion
module, the dental rinse comprising an effective amount of at least
one zinc chelator.
[0024] FIG. 1. (A) The five regions of Aap are illustrated: the
A-repeat region, the putative globular domain (.alpha./.beta.), the
B-repeat region containing 5 to 17 tandem G5 domains, the
collagen-like proline/glycine-rich region and a cell wall anchoring
motif (LPXTG). The proteolysis site is illustrated with scissors.
The domain boundaries of the Brpt1.5 and Brpt2.5 constructs are
illustrated, and the histidines are highlighted with arrowheads;
(B) Sequence alignment of the terminal G5 domain and C-terminal
"half repeat" motif from Aap (SEQ. ID NO. 1) and SasG (SEQ. ID NO.
2). Identical amino acids are indicated by dashes (-). Blast
alignment shows 80% conservation and 65% identity; (C) Far-UV
circular dichroism spectrum of Brpt1.5 in 20 mM Tris pH 7.4, 50 mM
NaF. Deconvolution of the data reveals predominantly .beta.-sheet
and coil secondary structure elements (Table 1); (D) Sedimentation
coefficient distribution plot for Brpt1.5 at varying
concentrations. Molecular weight estimation indicates Brpt1.5 is
monomeric; (E) Representative sedimentation equilibrium data for
Brpt1.5, confirming a monomeric state (Table 3). Black lines show
the global fits; residuals are shown above.
[0025] FIG. 2. (A) Sedimentation velocity analyses of Brpt1.5 in
the presence of divalent cations revealed a Zn.sup.2+-specific
dimerization event. The dashed line label (- - -) represents
cation-free Brpt1.5; (B) Far-UV CD spectrum of Brpt1.5 in the
presence (squares) and absence (triangles) of Zn; (C) Sedimentation
equilibrium analysis of Zn.sup.2+-mediated dimerization of Brpt1.5
(triangles) and Brpt2.5 (squares). Brpt2.5 dimerization requires
lower [Zn.sup.2+] (EC.sub.50=3.7 mM) compared to Brpt1.5
(EC.sub.50=5.4 mM) and shows a steeper slope for the monomer-dimer
transition, indicating enhanced cooperativity of Zn.sup.2+ binding
with increasing numbers of tandem G5 domains. Error bars show 95%
confidence intervals; (D) Linked equilibrium plot of Zn binding by
Brpt1.5. Linear regression analysis of the slope indicates that the
number of Zn.sup.2+ ions taken up upon Brpt1.5 dimerization (i.e.,
.DELTA.Zn.sup.2+) is approximately three; (E) Linked equilibrium
plot of Zn binding by Brpt2.5. Linear regression analysis of the
slope indicates that approximately five Zn.sup.2+ ions are bound
upon Brpt2.5 dimerization.
[0026] FIG. 3. (A) S. epidermidis RP62A biofilms formed on
polystyrene and were visualized with crystal violet; addition of
the Zn.sup.2+ chelator DTPA (.gtoreq.30 .mu.M) inhibits biofilm
formation. Representative wells have been scanned (bottom).
Untreated ("RP62A") and vehicle ("HCl") controls are shown. Error
bars show standard deviation (* indicates p<0.05 relative to
untreated control; n=3); (B) Methicillin-resistant S. aureus USA300
biofilms form on fibronectin-coated plates but are inhibited by
addition of DTPA (.gtoreq.30 .mu.M). (*: p<0.05; n=4); (C)
Addition of 5-20 .mu.M ZnCl.sub.2 at a minimal inhibitory dose of
DTPA (30 .mu.M) rescues biofilm formation by RP62A. (#: p<0.05
relative to the 0 .mu.M ZnCl.sub.2/30 .mu.M DTPA control; : data is
statistically indistinguishable from untreated control "RP62A";
n=3); (D) Addition of 15-20 .mu.M ZnCl.sub.2 at a minimal
inhibitory dose of DTPA (30 .mu.M) rescues biofilm formation by
USA300. (#: p<0.05 compared to 0 .mu.M ZnCl.sub.2/30 .mu.M DTPA;
: statistically indistinguishable from untreated control; n=4).
[0027] FIG. 4. (A) Addition of soluble MBP-Brpt1.5 inhibits RP62A
biofilms in a dose-dependent manner in the presence of 0.75-1 mM
ZnCl.sub.2. MBP alone was statistically indistinguishable from
untreated control at all concentrations tested. (*: p<0.05
relative to RP62A control at the relevant Zn.sup.2+ concentration;
n=3); (B) Dose response of biofilm inhibition by MBP-Brpt1.5 at a
fixed 1 mM ZnCl.sub.2 concentration. (*: p<0.0005; n=3); (C) The
"zinc zipper" model for intercellular adhesion in staphylococcal
biofilms mediated by zinc-dependent self-association of G5
domains.
[0028] FIG. 5. (A) Aap from S. epidermidis strain RP62A. Similar
architecture is observed in S. aureus proteins SasG and Pls, with
the reported number of G5 repeats across Staphylococcal species
varying from four to seventeen; (B) "Surface protein from gram
positive cocci" from Streptococcus suis 89/1591; (C) "LPXTG cell
wall surface protein" from Streptococcus gordonii str. challis
substr. ch1; (D) "Putative cell surface protein precursor" from
Corynebacterium urealyticum; (E). "Conserved hypothetical protein"
from Fingoldia magna; (F) .beta.-N-acetylhexosaminidase from
Streptococcus pneumoniae. Similar domain architecture is reported
in various S. pneumoniae strains; (G) IgA1 protease from S.
pneumoniae. Similar domain architecture is reported in predicted
zinc metalloproteases from S. pneumoniae, S. gordonii, and S. suis,
with up to four repeats of the G5 domain before the N-terminal
peptidase domain; (H). VanW vancomycin resistance protein from
Thermosinus carboxydivorans. Similar proteins are found in
Clostridium botulinum and C. difficile, Desulfotomaculum reducens,
Caldicellulosiruptor saccharoluticus, Pelotomaculum
thermopropionicum, Eubacterium ventriosum, Alkaliphilus sp.,
Thermonanaerobacter sp., Desulfitobacterium hafniense, Moorella
thermoacetica, Carboxydothermus hydreogenoformans, and
Symbiobacterium thermophilum.
[0029] FIG. 6. (A) Sedimentation velocity analysis of the
Zn.sup.2+-dependent dimerization of Brpt1.5 as a function of pH in
20 mM Tris, 20 mM MES, 50 mM NaCl, and 10 mM ZnCl.sub.2. Brpt1.5
shifts from dimer at pH 7.4 to monomer at pH 6.0; (B) Sedimentation
equilibrium confirms that Brpt1.5 transitions from dimer to monomer
as the pH is lowered from 7.4 to 6.0. The apparent pK.sub.a of this
transition is 6.7, which may indicate involvement of histidine
residues in Zn.sup.2+-dependent dimerization; (C) Linked
equilibrium analysis of the pH dependence of Brpt1.5 dimerization
at 10 mM ZnCl.sub.2. The slope of the logK versus log [H.sup.+]
plot, determined by linear regression, reveals that Brpt1.5
dimerization is linked to the release of approximately 2 or 3
protons; (D) Far-UV CD spectrum of the H75A/H85A Brpt1.5 mutant in
the presence (squares) and absence (triangles) of 10 mM ZnCl.sub.2;
(E) Sedimentation velocity analysis of wild-type Brpt1.5 (gray)
compared to histidine-null mutant H75A/H85A (black). Dashed lines
show data collected in the absence of Zn.sup.2+, and solid lines
show data collected at 10 mM ZnCl.sub.2. Partial loss of
dimerization is observed in the H75A/H85A mutant, suggesting that
one or both histidines as well as other residues are involved in
Zn.sup.2+ coordination.
[0030] FIG. 7. (A) SrCl.sub.2 does not reverse biofilm inhibition
by 30 .mu.M DTPA. All data at 30 .mu.M DTPA show statistically
significant decreases compared to no-treatment control (RP62A); (B)
CaCl.sub.2 does not reverse biofilm inhibition by 30 .mu.M DTPA.
All data at 30 .mu.M DTPA show statistically significant decreases
compared to no-treatment control (RP62A); (C) MgCl.sub.2 does not
reverse biofilm inhibition by 30 .mu.M DTPA. All data at 30 .mu.M
DTPA show statistically significant decreases compared to
no-treatment control (RP62A); (D) Addition of 10 .mu.M or greater
MnCl.sub.2 effected partial rescue of biofilm formation in the
presence of 30 .mu.M DTPA. All data at 30 .mu.M DTPA show
statistically significant decreases compared to no-treatment
control (RP62A). Statistically significant increases over the 0
.mu.M MnCl.sub.2/30 .mu.M DTPA datapoint are marked with an
asterisk (*). The levels of MnCl.sub.2 required to rescue biofilm
formation were at least 500 times higher than the concentration of
Mn in serum (.about.20 nM), indicating that this is unlikely to be
a biologically relevant phenomenon. This result may be due to
competition for DTPA and release of chelated zinc. The affinity of
Mn for DTPA (log K=15.5) is within approximately three orders of
magnitude of the Zn affinity (log K=18.75), whereas the affinities
of Ca, Sr, and Mg for DTPA are 8-9 orders of magnitude lower than
zinc (log K=10.74, 9.68, and 9.3, respectively).
[0031] FIG. 8. Inhibition of RP62A biofilms by MBP-Brpt1.5 at 750
.mu.M ZnCl.sub.2. Dose response of biofilm inhibition by
MBP-Brpt1.5 at a fixed 750 .mu.M ZnCl.sub.2 concentration.
Asterisks indicate the level of statistical significance relative
to the untreated control.
[0032] The following is a list of definitions for terms used
herein.
[0033] The term "biofilm" refers to matrix-enclosed microbial
accretions to biological or non-biological surfaces. Biofilm
formation represents a protected mode of growth that allows cells
to survive in hostile environments.
[0034] The term "biofilm formation" is intended to include the
formation, growth, and modification of the bacterial colonies
contained with biofilm structures, as well as the synthesis and
maintenance of the polysaccharide matrix of the biofilm
structures.
[0035] The term "zinc adhesion module" refers to a polypeptide fold
found in bacterial cell-surface proteins including, but not limited
to, the Accumulation-associated protein (Aap) from Staphylococcus
epidermidis. The zinc adhesion module comprises a polypeptide
sequence that includes at least one G5 domain, and optionally
additional amino acid sequence.
[0036] The term "G5 domain" refers to a polypeptide fold found in a
wide variety of extracellular proteins, named after its conserved
glycine residues. The G5 domain consists of approximately 80 amino
acid residues and is typically found as one to twelve tandem
repeats. The G5 domain is the first 80 amino acids of Brpt1.0.
[0037] The terms "B-repeat" or "Brpt" refer to a region of the Aap
protein having a variable number (5 to 17) of nearly identical 128
amino acid repeats. "Brpt1.5" refers to a B-repeat region
terminating in a conserved 79 amino acid "half repeat" motif.
"Brpt1.0" refers to an isolated 128 amino acid repeat comprising a
G5 domain.
[0038] The terms "chelator" or "metal chelator" refer to any
substance that is able to remove a metal ion from a solution system
by forming a new complex ion that has different chemical properties
than those of the original metal ion. The term is further intended
to encompass substances that are capable of chelating metal ions,
specifically divalent metals.
[0039] The term "metal ions" is intended to include any metal ion
that is bioavailable, i.e., any metal ion involved in a biochemical
reaction or pathway, or any metal ion that is available in the
fluid, tissue, or bone of a subject.
[0040] The term "zinc chelator" refers to any substance that is
able to chelate a zinc (Zn.sup.2+) ion and thus deplete zinc from
aqueous environments.
[0041] The term "gram positive bacteria" refers to bacteria having
cell walls with high amounts of peptidoglycan. Gram positive
bacteria are identified by their tendency to retain crystal violet
and stain dark blue or violet in the Gram staining protocol.
[0042] The term "gram negative bacteria" refers to bacteria having
thinner peptidoglycan layers which do not retain the crystal violet
stain in the Gram staining protocol and instead retain the
counterstain, typically safranin. Gram negative bacteria stain red
or pink in the Gram staining protocol.
[0043] The term "implantable medical device" refers to any medical
device implanted or inserted in the human body. Such devices can be
temporarily or permanently implanted or inserted. An implantable
medical device can be, for example, catheters, orthopedic devices,
prosthetic devices, vascular stents, urinary stents, pacemakers,
implants, or the like.
[0044] The term "bathing a device" refers to submerging a device in
a solution in order to pre-treat the device, for example, prior to
surgical implantation. Bathing a device can also occur after the
device has been surgically implanted, for example, by irrigating
the surgical site with a sterile solution.
[0045] The term "coating a device" refers to pre-treating a device
with a composition prior to surgical implantation. Suitable
compositions for pre-treating the device may include, for example,
solutions, gels, polymer coatings, and the like. A variety of means
may be employed to coat a device, such as spraying or submerging
the device. The coated device comprises a surface layer having
desirable properties conferred by the coating composition. In one
embodiment, the coating composition comprises at least one zinc
chelator. In another embodiment, the coating composition comprises
one or more soluble G5 domains or zinc adhesion modules.
[0046] The term "polymer coating" refers to an adherent polymer
layer suitable for coating the exterior surface of a device, for
example, an implantable medical device. The polymer coating may
have intrinsic chelation properties, or may be bonded to one or
more chemical or protein-based chelating agents.
[0047] The term "mammal" refers to organisms that can suffer from
biofilm-associated states. The term includes humans, for example,
as well as wild and domesticated animals and livestock, including
but not limited to horses, chimpanzees, macaques, pigs, sheep,
goats, hamsters, guinea pigs, monkeys, bears, dogs, cats, mice,
rabbits, cattle, squirrels, and rats. The term "pharmaceutical
composition" includes preparations suitable for administration to
mammals, for example, humans.
[0048] The term "topical pharmaceutical composition" refers to
pharmaceutical compositions suitable for dermal administration to a
mammal. Suitable topical pharmaceutical compositions include, but
are not limited to, gels, creams, lotions, ointments, tinctures,
sprays, and solids. In one embodiment, a topical pharmaceutical
composition of the present invention is applied on the outer
surface of the skin or in the vicinity of cuts, abrasions, turf
burn injuries, lacerations, burns, or puncture wounds in order to
treat, prevent, or inhibit the formation of bacterial biofilms.
[0049] The term "antimicrobial agent" refers to any substance that
kills or prevents the growth of bacteria or other microbes.
[0050] The term "antibiotic" refers to a substance that is
antagonistic to the growth of microorganisms. Suitable antibiotics
may be naturally-occurring, chemically-modified, or
synthetically-produced.
[0051] The term "surgical rinse" refers to a solution used during
surgery to irrigate the site of an implanted medical device, with
the intent to prevent initial formation of biofilms in the vicinity
of the medical device.
[0052] The term "dental rinse" refers to a solution containing one
or more zinc chelators used as a mouthwash or rinse to prevent the
establishment of oral biofilms that lead to dental caries.
[0053] The term "personal cleansing composition" refers to a
composition that is used for personal hygiene. Personal cleansing
compositions include, but are not limited to, gels, creams,
suspensions, colloids, soaps, body washes, shampoos, and the like.
In one embodiment, the personal cleansing compositions of the
present invention inhibit biofilm-related infections including, but
not limited to, community-acquired methicillin-resistant S. aureus
(CA-MRSA) infection.
[0054] The term "hard surface cleaning composition" refers to a
composition that is used to clean and/or sanitize a hard or solid
surface. In one embodiment, the invention provides a composition
that prevents bacterial biofilm growth on hard surfaces including,
but not limited to, surgical instruments, storage tanks, pipelines,
trays, containers, walls, floors, countertops, locker room floors,
benches, lockers, showers, bathrooms, toilets, water filtration
units, and the like.
[0055] The terms "inhibit," "inhibiting," and "inhibited" as used
herein with respect to biofilm formation, refer to the effect of a
zinc chelator in disrupting or clearing a biofilm, as well as
preventing formation of a biofilm.
[0056] The term "effective amount," as used herein with respect to
inhibiting biofilm formation, refers to an amount of a zinc
chelator or a soluble zinc adhesion module sufficient to achieve
the desired inhibitory result.
[0057] The term "safe and effective amount" refers to an amount of
a zinc chelator or a soluble zinc adhesion module that is effective
to inhibit biofilm formation without undue adverse side effects,
such as toxicity, irritation, or allergic response, commensurate
with a reasonable risk/benefit ration when used in the manner of
the invention.
[0058] The term "therapeutically effective amount" refers to a
sufficient amount of an ingredient to treat disorders, at a
reasonable benefit/risk ratio applicable to any medical treatment.
It will be understood, however, that the total daily usage of the
compositions of the present invention will be decided by the
attending physician within the scope of sound medical judgment. The
specific therapeutically effective dose level for any particular
patient will depend upon a variety of factors including the
disorder being treated and the severity of the disorder; activity
of the specific chelator employed; the specific composition
employed; the age, body weight, general health, sex and diet of the
patient; the time of administration, route of administration, and
rate of excretion of the specific compound employed; the duration
of the treatment; drugs used in combination or coincidental with
the specific compound employed; and like factors well known in the
medical arts. For example, it is well within the skill of the art
to start doses of the compound at levels lower than required to
achieve the desired therapeutic effect and to gradually increase
the dosage until the desired effect is achieved.
Solution Characterization of the Terminal G5 Domain from Aap
[0059] The present invention indicates that homophilic association
of Aap and SasG is mediated solely via self-association of their
respective G5 domains. The C-terminal G5 domain from the B-repeat
region of Aap was expressed both as an isolated domain (called
Brpt1.0) and as the single G5 domain followed by the C-terminal 79
amino acid "half-repeat" (called Brpt1.5; FIGS. 1A and 1B).
[0060] This C-terminal half-repeat is conserved in Aap homologs,
suggesting that it may function as a terminal cap required for the
stability of the B-repeat region, as seen with other proteins
containing repeating structural motifs. Solution characterization
of Brpt1.0 by far-UV circular dichroism (CD) showed a preponderance
of random coil with little evidence of regular structural elements.
In contrast, Brpt1.5 samples yielded consistent spectra
corresponding to 39% .beta.-sheet, 32% coil, 24% turn, and 4%
.alpha.-helix (FIG. 1C, Table 1).
TABLE-US-00001 TABLE 1 Circular dichroism secondary structure
analyses of Brpt1.5 % .alpha.-helix % .beta.-sheet % Turn % Coil
Brpt1.5 4 39 24 32 Brpt1.5 + 10 mM ZnCl.sub.2 5 38 23 32 H75A/H85A
4 38 23 32 H75A/H85A + 10 mM ZnCl.sub.2 5 40 22 32 Far-UV CD
spectra were collected on samples dialyzed into 20 mM Tris, 50 mM
NaF. Secondary structure content was determined using the program
CDSSTR on Dichroweb. NRMSD values ranged from 0.039 to 0.043.
[0061] These data support the role of the half repeat as a
structural cap required for stability of the G5 domain.
Sedimentation velocity analytical ultracentrifugation (AUC)
experiments revealed that Brpt1.5 sedimented as a single elongated
species with an estimated molecular weight consistent with a
monomer (FIG. 1D, Table 2).
TABLE-US-00002 TABLE 2 Sedimentation velocity parameters for
Brpt1.5 s.degree..sub.20, w f/f.sub.0 Brpt1.5 1.55 2.21 Brpt1.5 +
10 mM ZnCl.sub.2 2.46 1.66 Values of s.degree..sub.20, w were
determined from data at 4 or 5 concentrations. Values of the
frictional ratio, f/f.sub.0, were averaged from four or more
independent experiments.
[0062] No assembly to a higher order species was observed under
these conditions, even at high (70 .mu.M) concentrations.
Sedimentation equilibrium experiments verified that Brpt1.5 was
monomeric under these conditions (FIG. 1E, Table 3).
TABLE-US-00003 TABLE 3 Sedimentation equilibrium parameters for
Brpt1.5 and Brpt2.5 plus ZnCl.sub.2. M.sub.sequence
M.sub.experimental (95% confidence) monomer dimer 0 mM Zn 10 mM Zn
Brpt1.5 22,284 44,568 22,075 41,213 (19,820-24,259) (38,823-43,536)
Brpt2.5 35,971 71,942 36, 987 78,541 (34,519-39,380)
(76,220-80,863) K.sub.D for monomer-dimer association 2 mM Zn 5 mM
Zn 10 mM Zn Brpt1.5 113 .mu.M 5.06 .mu.M 0.353 .mu.M (62.8-200
.mu.M) (3.59-7.08 .mu.M) (0.196-0.647 .mu.M) Brpt2.5 25.5 .mu.M
0.072 .mu.M ND* (17.8-36.8 .mu.M) (0.165-0.0322 .mu.M) *ND, Not
determined; no detectable monomer was observable under this
condition, precluding the determination of an equilibrium constant.
95% confidence intervals calculated by WinNONLIN. M.sub.sequence is
the molecular weight determined by ProtParam from the amino acid
sequence. M.sub.experimental is determined from global analysis of
sedimentation equilibrium curves.
Zn.sup.2+-Mediated Dimerization of G5 Domains
[0063] Since many mammalian adhesion proteins including cadherins,
integrins, and neurexins require divalent cations for proper
function, Brpt1.5 was analyzed by AUC in the presence of Ca.sup.2+,
Sr.sup.2+, Mg.sup.2+, Mn.sup.2+, Ni.sup.2+, Co.sup.2+, and
Zn.sup.2+. Interestingly, Brpt1.5 formed dimers only in the
presence of Zn.sup.2+ (FIG. 2A, Tables 2 and 3). Sedimentation
equilibrium analyses of Brpt1.5 in the presence of 2, 5, or 10 mM
ZnCl.sub.2 revealed monomer-dimer equilibria with dissociation
constants of 113 .mu.M, 5.06 .mu.M, and 0.353 .mu.M, respectively
(Table 3). CD spectra of Brpt1.5 in the presence or absence of 10
mM ZnCl.sub.2 were similar, indicating that Zn.sup.2+-mediated
Brpt1.5 dimerization occurs as a result of rigid-body association
of two G5 domains mediated through coordination of one or more
Zn.sup.2+ ions in trans rather than significant Zn.sup.2+-induced
conformational changes (FIG. 2B, Table 1).
[0064] In Aap and the related staphylococcal proteins SasG and Pls,
G5 domains are found as tandem repeats ranging from 5 to 17 copies.
SasG-dependent protein-based biofilm formation in S. aureus has
been shown to require at least five tandem G5 repeats. See
Corrigan, Microbiology 153: 2435-46. To analyze the behavior of
tandem G5 domains, a Brpt2.5 construct with two G5 domains and the
C-terminal cap was expressed.
[0065] Sedimentation equilibrium analysis of Brpt2.5 revealed a
monomer-dimer equilibrium in the presence of Zn.sup.2+ as observed
for Brpt1.5, but with a modest enhancement in Zn.sup.2+ affinity
for Brpt2.5 compared to Brpt1.5, combined with a steeper transition
between monomer and dimer as the repeat length increased (FIG. 2C).
To analyze the linked equilibria between dimerization and Zn.sup.2+
binding, the logarithm of the dimerization association constant was
plotted against the logarithm of the free Zn.sup.2+ concentration
to reveal the net number of Zn.sup.2+ ions participating in the
dimerization interface. There are 2-3 Zn.sup.2+ ions participating
in the Brpt1.5 dimer interface, compared to .about.5 Zn.sup.2+ ions
for Brpt2.5. This is consistent with a modular arrangement in which
each G5 domain contributes to an independent dimer interface (FIGS.
2D and 2E).
Zn.sup.2+ Coordination by the G5 Domain
[0066] Structural Zn.sup.2+ ions are most commonly coordinated by
cysteine, followed by histidine, aspartate and glutamate. The
entire sequence of Aap lacks cysteines; however, there are two
histidine residues in the Brpt1.5 construct. Brpt1.5 with 10 mM
Zn.sup.2+ transitions from a dimer at pH 7.4 to a monomer at pH 6.0
(FIG. 6A). Sedimentation equilibrium experiments verified these
results; a plot of the relative molecular weight as a function of
pH could be fitted to yield an apparent pK.sub.a of 6.7 (FIG. 6B),
consistent with coordination by histidine. Linear regression
analysis of the linked equilibria between protonation and
dimerization by use of a double-log plot showed a .DELTA.H.sup.+
value of 2.6, indicating that approximately three ionizable
residues are linked to dimerization of Brpt1.5 (FIG. 6C).
Site-directed mutagenesis confirmed the involvement of histidines,
although other residues such as glutamates or aspartates are likely
to contribute as well (FIGS. 6D and 6E). Taken together, the CD and
AUC results for Brpt1.5 and Brpt2.5 indicate that the G5 domains of
Aap self-assemble in a modular fashion upon Zn.sup.2+ binding. The
presence of tandem domain repeats and dependence upon a divalent
cation for adhesion are reminiscent of calcium-dependent adhesion
by mammalian cadherins.
Zn.sup.2+ Chelation Inhibits Staphylococcal Biofilm Formation
[0067] To test the physiological role of Zn.sup.2+-mediated G5
adhesion in the formation of staphylococcal biofilms, both S.
epidermidis and S. aureus biofilms were grown in culture in the
presence or absence of the Zn.sup.2+ chelator
diethylenetriaminepentaacetic acid (DTPA). DTPA was non-toxic at
all concentrations tested (up to 100 .mu.M). S. epidermidis strain
RP62A formed robust biofilms when cultured in media alone, but
biofilm growth was inhibited by non-toxic doses of DTPA (.gtoreq.30
.mu.M) (FIG. 3A). Under the conditions tested, DTPA did not disrupt
pre-formed RP62A biofilms.
[0068] S. aureus strain USA300, which is responsible for recent
epidemic outbreaks of community-acquired methicillin-resistant S.
aureus (MRSA) infections, formed biofilms on fibronectin-coated
plates. As observed for S. epidermidis, the MRSA biofilms were
inhibited by Zn.sup.2+ chelation (.gtoreq.30 .mu.M DTPA) (FIG. 3B).
Surprisingly, S. epidermidis biofilm formation could be rescued by
the addition of 5-20 .mu.M ZnCl.sub.2, indicating that the effect
is Zn.sup.2+-specific (FIG. 3C). Addition of up to 60 .mu.M
CaCl.sub.2, MgCl.sub.2, and SrCl.sub.2 had no effect; MnCl.sub.2
effected partial rescue, but at concentrations nearly 1,000-fold
above physiological levels (FIG. 7). Like S. epidermidis, the MRSA
strain USA300 could be rescued by the addition of 15-20 .mu.M
ZnCl.sub.2 (FIG. 3D). Importantly, at the minimal inhibitory
concentration of DTPA (30 .mu.M), biofilm growth by both
staphylococcal species could be rescued by the addition of 5-20
.mu.M ZnCl.sub.2; these Zn.sup.2+ concentrations are similar to the
physiological range in human plasma (12-16 .mu.M resting
concentration).
Soluble G5 Domain Inhibits Biofilm Formation
[0069] To determine whether Zn.sup.2+-dependent biofilm formation
is specifically dependent upon G5 domain-mediated self-assembly,
soluble Brpt1.5 was added to S. epidermidis RP62A biofilms as a
maltose-binding protein (MBP) fusion. Because of the higher
Zn.sup.2+ concentrations required to induce dimerization in the
short construct compared to full-length Aap, inhibition by
MBP-Brpt1.5 was tested at Zn.sup.2+ concentrations ranging up to 1
mM. RP62A biofilms were inhibited by soluble MBP-Brpt1.5 in the
presence of 0.75 and 1 mM ZnCl.sub.2, whereas addition of MBP alone
had no effect (FIG. 4A). Titration of MPB-Brpt1.5 at fixed
Zn.sup.2+ concentrations showed that at 1 mM ZnCl.sub.2, addition
of 10-22 .mu.M MBP-Brpt1.5 completely abolished biofilm formation
(FIG. 4B). Dose-dependent biofilm inhibition by MBP-Brpt1.5 also
was observed at 750 .mu.M ZnCl.sub.2 (FIG. 8). Thus, addition of
even a single intact zinc adhesion module in a soluble form is
capable of inhibiting biofilm formation, highlighting the role of
this domain as a Zn.sup.2+-dependent adhesion module in
staphylococcal biofilms.
[0070] The G5 domain, found in Aap, SasG, and related
staphylococcal proteins such as Pls, mediates Zn.sup.2+-dependent
staphylococcal biofilm formation. Multiple self-association events
between stretches of tandem G5 domains in opposing Aap molecules
would lead to an extensive adhesive contact between two cells, a
so-called "zinc zipper" (FIG. 4C). Aap was originally identified as
a protein required for the formation of S. epidermidis
microcolonies. Subsequently, Aap and SasG were shown to undergo a
proteolysis event near the beginning of the B-repeat region
containing the tandem G5 domains, which leads to formation of
protein-based staphylococcal biofilms. The present invention
indicates that the same mechanism for intercellular adhesion based
upon Zn.sup.2+-dependent G5 self-association is responsible for
early and late stages of biofilm formation. Zn.sup.2+-dependent
intercellular adhesion in biofilms mediated by G5 domains has
likely evolved as a defense mechanism against immune cell action.
Zn.sup.2+ levels that are known to increase cytokine release and
boost host immune responses are sufficient to promote
staphylococcal biofilm formation (5-20 .mu.M Zn.sup.2+). Mast
cells, basophils, and eosinophils contain high levels of Zn.sup.2+
in secretory granules that is released upon degranulation.
[0071] Given the global emergence of staphylococcal biofilm-related
infections, improved methods for prevention are needed. Targeting
G5 self-association either by Zn.sup.2+ chelation or direct
inhibition with small molecule compounds provides a new therapeutic
avenue for combating the formation of biofilms by a broad range of
bacterial species, including epidemic MRSA strains. The application
to MRSA infections is of particular interest, given that MRSA was
recently reported to cause more deaths per year than AIDS. King, M.
D. et al, Emergence of community-acquired methicillin-resistant
Staphylococcus aureus USA 300 clone as the predominant cause of
skin and soft-tissue infections, Ann. Intern. Med. 144:309-17
(2006). Finally, genes encoding surface proteins with one or more
tandem G5 domains are found in a vast number of bacterial species,
indicating that the zinc zipper mechanism for intercellular
adhesion provides a target for inhibition of biofilm formation
across multiple bacterial species, in both medical and industrial
applications.
Biofilm-Forming Bacteria
[0072] For each of the subsequent embodiments of the invention, the
biofilm-forming bacteria comprise at least one zinc adhesion
module, such that contact with one or more zinc chelators or one or
more soluble zinc adhesion modules inhibits formation of a biofilm.
For each embodiment, the bacteria comprising at least one zinc
adhesion module may be selected from the group consisting of all
gram-positive bacteria.
[0073] More specifically, the bacteria for each of the subsequent
embodiments may be selected from the group consisting of
Acidothermus cellulyticus, Actinomyces odontolyticus, Alkaliphilus
metalliredigens, Alkaliphilus oremlandii, Arthrobacter aurescens,
Bacillus amyloliquefaciens, Bacillus clausii, Bacillus halodurans,
Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis,
Bifidobacterium adolescentis, Bifidiobacterium longum,
Caldicellulosiruptor saccharolyticus, Carboxydothermus
hydrogenoformans, Clostridium acetobutylicum, Clostridium
beijerinckii, Clostridium botulinum, Clostridium cellulolyticum,
Clostridium difficile, Clostridium kluyveri, Clostridium leptum,
Clostridium novyi, Clostridium perfringens, Clostridium tetani,
Clostridium thermocellum, Corynebacterium diphtheriae,
Corynebacterium efficiens, Corynebacterium glutamicum,
Corynebacterium jeikeium, Corynebacterium urealyticum,
Desulfitobacterium hafniense, Desulfotomaculum reducens,
Eubacterium ventriosum, Exiguobacterium sibiricum, Fingoldia magna,
Geobacillus kaustophilus, Geobacillus thermodenitrificans,
Janibacter sp., Kineococcus radiotolerans, Lactobacillus fermentum,
Listeria monocytogenes, Listeria innocua, Listeria welshimeri,
Moorella thermoacetica, Mycobacterium avium, Mycobacterium bovis,
Mycobacterium gilvum, Mycobacterium leprae, Mycobacterium
paratuberculosis, Mycobacterium smegmatis, Mycobacterium
tuberculosis, Mycobacterium ulcerans, Mycobacterium vanbaalenii,
Nocardioides sp., Nocardia farcinica, Oceanobacillus iheyensis,
Pelotomaculum thermopropionicum, Rhodococcus sp., Saccharopolyspora
erythraea, coagulase-negative Staphylococcus species,
Staphylococcus aureus, methicillin resistant Staphylococcus aureus
(MRSA), Staphylococcus epidermidis, methicillin resistant
Staphylococcus epidermidis (MRSE), Streptococcus agalactiae,
Streptococcus gordonii, Streptococcus mitis, Streptococcus oralis,
Streptococcus pneumoniae, Streptococcus sanguinis, Streptococcus
suis, Streptomyces avermitilis, Streptomyces coelicolor,
Thermoanaerobacter ethanolicus, Thermoanaerobacter tengcongensis,
and combinations thereof.
[0074] Even more specifically, the bacteria for each of the
subsequent embodiments may be selected from the group consisting of
Corynebacterium urealyticum, Fingoldia magna, Staphylococcus
aureus, methicillin resistant Staphylococcus aureus (MRSA),
Staphylococcus epidermidis, methicillin resistant Staphylococcus
epidermidis (MRSE), Streptococcus gordonii, Streptococcus
pneumoniae, Streptococcus sanguinis, Streptococcus suis, and
combinations thereof.
[0075] More specifically still, the bacteria for each of the
subsequent embodiments may be selected from the group consisting of
Staphylococcus aureus, methicillin resistant Staphylococcus aureus
(MRSA), Staphylococcus epidermidis, methicillin resistant
Staphylococcus epidermidis (MRSE), and combinations thereof.
[0076] It has also been discovered that certain gram negative
bacteria comprise at least one zinc adhesion module. For example,
gram negative bacteria comprising a zinc adhesion module include,
but are not limited to, Bacteroides capillosus, Symbiobacterium
thermophilum, Syntrophomonas wolfei, Thermosinus carboxydivorans.
Accordingly, each of the subsequent embodiments of the invention
may also be applied to gram negative bacteria having at least one
zinc adhesion module.
Zinc Chelators
[0077] One skilled in the art will appreciate the variety of
substances capable of chelating zinc metal ions and will select an
appropriate chelator for use in the particular embodiment of the
invention. For each of the subsequent embodiments of the invention,
the zinc chelator may selected from the group consisting of
ethylenediaminetetra-acetic acid (EDTA),
1,3-diaminopropane-N,N,N',N'-tetraacetic acid (DTPA),
N,N,N',N'-tetrakis(2-pyrdiylmethyl)ethylenediamine (TPEN),
1,10-phenanthroline, clioquinol, diethyldithiocarbamate (DEDTC),
2,3-dimercapto-1-propanesulfonic acid (DMPS),
ethylenediamine-N,N'-diacetic-N,N'-di-B-propionic acid (EDPA),
1,2-dimethyl-3-hydroxy-4-pyridinone (DMHP),
1,2-diethyl-3-hydroxy-4-pyridinone (DEHP), ethylmaltol (EM),
4-(6-Methoxy-8-quinaldinyl-aminosulfonyl)benzoic acid potassium
salt (TFLZn), dithiozone,
N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ), carnosine,
deferasirox, trans-1,2-cyclohexane-diamine-N,N,N',N'-tetraacetic
acid (CyDTA), dihydroxyethylglycine (DHEG),
1,3-diamino-2-hydroxypropane-N,N,N',N'-tetraacetic (DTPA-OH),
ethylenediamine-N,N'-diacetic acid (EDDA),
ethylenediamine-N,N'-dipropionic acid (EDDP),
ethylenediamine-N,N'-bis(methylphosphonic) acid (EDDPO),
N-hydroxy-ethylenediamine-N,N',N'-triacetic acid (EDTA-OH),
ethylenediaminetetra(methylenephosphonic) acid (EDTPO),
N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED),
hexamethylene-1,6-diaminetetraacetic acid (HDTA),
hydroxyethyliminodiacetic acid (HIDA), iminodiacetic acid (IDA),
Methyl-EDTA, nitrilotriacetic acid (NTA), nitrilotripropionic acid
(NTP), Nitrilotrimethylenphosphonic acid (NTPO),
7,19,30-trioxa-1,4,10,13,16,22,27,33-octaazabicyclo[11,11,11]pentatriacon-
tane (O-Bistren), triethylenetetraaminehexaacetic acid (TTHA),
ethyleneglycol bis(2-Aminoethyl ether)-N,N,N',N'-tetraacetic acid
(EGTA), dimercaptosuccinic acid (DMSA), deferoxamine, dimercaprol,
zinc citrate, combination of bismuth and citrate, penicilamine,
succimer, Etidronate, ethylenediamine-di(O-hydroxyphenylacetic
acid) (EDDHA), trans-1,2-cyclohexanediaminetetraacetic acid (CDTA),
N-(2-hydroxyethyl)ethylenedinitrilotriacetic acid (HEDTA),
N-(2-hydroxyethyl)iminodiacetic acid (HEIDA), calprotectin, zinc
fingers, lactoferrin, ovotransferrin, conalbumin, and combinations
thereof.
[0078] More specifically, for each of the subsequent embodiments,
the zinc chelator may be selected from the group consisting of
DTPA, TPEN, 1,10 phenanthroline, EDTA, DEDTC, EDDA and combinations
thereof. Still more specifically, for each of the subsequent
embodiments, the zinc chelator can be DTPA.
[0079] In one embodiment of the invention, a method is provided for
inhibiting formation of a biofilm comprising bacteria, the method
comprising contacting the bacteria with an effective amount of at
least one zinc chelator, wherein the bacteria comprise at least one
zinc adhesion module and wherein formation of the biofilm is
inhibited.
[0080] In one aspect of the embodiment, the at least one zinc
chelator is placed in contact with the bacteria within an
environment that supports bacterial adhesion and therefore
potential establishment of biofilm formation. Bacterial zinc
deprivation at the site of adhesion prevents zinc adhesion module
interaction and, accordingly, protein-dependent biofilm formation.
Zinc chelators, however, are not necessarily bactericidal and may
not directly kill cells, but instead prevent establishment of
infection.
[0081] A variety of zinc chelators are known in the art and may be
employed in the present embodiment. Determining the appropriate
concentration of the zinc chelator, based on its properties and the
targeted bacterial biofilm(s), is within the purview of the skilled
artisan. For example, appropriate concentrations of DTPA for use in
the present embodiment against the bacteria Staphylococcus aureus,
methicillin resistant Staphylococcus aureus, Staphylococcus
epidermidis, methicillin resistant Staphylococcus epidermidis,
Streptococcus gordonii, Streptococcus pneumoniae, Streptococcus
sanguinis, and Streptococcus suis may range from about 10 .mu.M to
about 1000 .mu.M, from about 10 .mu.M to about 800 .mu.M, from
about 10 .mu.M to about 700 .mu.M, from about 20 .mu.M to about 640
.mu.M, from about 20 .mu.M to about 500 .mu.M, from about 20 .mu.M
to about 400 .mu.M, from about 20 .mu.M to about 320 .mu.M, from
about 20 .mu.M to about 200 .mu.M, or from about 20 .mu.M to about
160 .mu.M.
[0082] In another embodiment of the invention, a method is provided
for inhibiting formation of a biofilm on a device, wherein the
biofilm comprises bacteria comprising at least one zinc adhesion
module, the method comprising contacting the device with a solution
comprising an effective amount of at least one zinc chelator,
wherein formation of a biofilm on the device is inhibited.
[0083] In one aspect of the embodiment, the device is an
implantable medical device. One skilled in the art will appreciate
that any medical device implanted or inserted into the body is
suitable for use in the present embodiment. For example, suitable
implantable medical devices include, but are not limited to,
pacemakers, heart valves, replacement joints, catheters, catheter
access ports, dialysis tubing, gastric bands, shunts, screw plates,
artificial spinal disc replacements, internal implantable
defibrillators, cardiac resynchronization therapy devices,
implantable cardiac monitors, mitral valve ring repair devices,
left ventricular assist devices (LVADs), artificial hearts,
implantable infusion pumps, implantable insulin pumps, stents,
implantable neurostimulators, maxillofacial implants, dental
implants, and the like.
[0084] The biofilm-forming bacteria of the embodiment comprise at
least one zinc adhesion module, such that contact with one or more
zinc chelators inhibits formation of a biofilm on the device.
[0085] Determining the appropriate concentration of the zinc
chelator, based on its properties and the bacterial biofilm at
issue, is well within the purview of the skilled artisan. In one
embodiment, the concentration of the chelator can be from about 20
.mu.M to about 3 mM. For example, appropriate concentrations of
DTPA for use in the present method with S. epidermidis RP62A may
range from about 20 .mu.M to about 2.6 mM, or more specifically
from about 20 .mu.M to about 80 .mu.M, or still more specifically
from about 30 .mu.M to about 80 .mu.M; with S. aureus USA300, from
about 80 .mu.M to about 1300 .mu.M, or more specifically from about
80 .mu.M to about 1600 .mu.M with S. aureus SA113, from about 10
.mu.M to about 160 .mu.M, or more specifically from about 10 .mu.M
to about 30 .mu.M and with S. aureus COL, from about 10 .mu.M to
about 160 .mu.M, or more specifically from about 10 .mu.M to about
30 .mu.M.
[0086] In one aspect of the embodiment, the device is bathed or
coated in a solution comprising an effective amount of at least one
zinc chelator. For example, the device may be dipped in a solution
comprising an effective amount of a zinc chelator and optionally
allowed to dry. In another aspect of the embodiment, the device may
be sprayed with a solution comprising a zinc chelator and
optionally allowed to dry. In a surgical setting, the present
method could be used, for example, to bathe a medical device prior
to implantation. Alternatively, a medical device could be coated
with a solution comprising a zinc chelator prior to
implantation.
[0087] The solution comprising the zinc chelator can be in any form
which permits application of the solution to the particular device.
In a specific embodiment, the solution is a gel comprising at least
one zinc chelator. In another specific embodiment, the solution is
a polymer coating comprising at least one zinc chelator.
Optionally, the solution may be composed such that an effective
amount of a zinc chelator is released gradually, providing for
inhibition of biofilm formation over a period of time.
[0088] In another embodiment of the invention, a topical
pharmaceutical composition for inhibiting formation of a biofilm in
or on a mammal is provided, wherein the biofilm comprises bacteria
comprising at least one zinc adhesion module, the pharmaceutical
composition comprising a therapeutically effective amount of at
least one zinc chelator and at least one pharmaceutically
acceptable carrier.
[0089] The phrase "pharmaceutically acceptable carrier" is art
recognized and includes a pharmaceutically acceptable material,
composition or vehicle, suitable for administering compositions of
the present invention to mammals. Each carrier must be "acceptable"
in the sense of being compatible with the other ingredients of the
formulation and not injurious to the patient.
[0090] The compositions of this invention can be administered
topically to a subject, e.g., by the direct laying on or spreading
of the composition on the epidermal or epithelial tissue of the
subject. Such compositions include, for example, lotions, creams,
solutions, gels, sprays, ointments, and solids. These topical
compositions preferably comprise a therapeutically effective
amount, usually at least about 20 .mu.M, and specifically from
about 40 .mu.M to about 2 mM, of a zinc chelator. Suitable carriers
for topical administration preferably remain in place on the skin
as a continuous film, and resist being removed by perspiration or
immersion in water. Generally, the carrier is organic in nature and
capable of having dispersed or dissolved therein at least one zinc
chelator. The carrier may include pharmaceutically-acceptable
emollients, emulsifiers, thickening agents, solvents, and the
like.
[0091] The amount of zinc chelator to be topically administered
depends upon such factors as skin sensitivity, type and location of
the tissue to be treated, the composition and carrier to be
administered, the particular zinc chelator to be administered, as
well as the particular bacterial biofilm to be inhibited.
[0092] The compositions of the invention may further include
additional drugs or excipients as appropriate for the indication.
In one aspect of the embodiment, the pharmaceutical composition
further comprises a therapeutically effective amount of at least
one antimicrobial agent. In a more specific aspect, the
antimicrobial agent is an antibiotic.
[0093] In another embodiment of the invention, a surgical rinse for
inhibiting the formation of a biofilm is provided, wherein the
biofilm comprises bacteria comprising at least one zinc adhesion
module, and wherein the surgical rinse comprises an effective
amount of at least one zinc chelator. In one embodiment, the
surgical rinse may be, for example, a buffered saline solution or a
Ringer's solution. In another embodiment, the chelator is DTPA and
the concentration can be from about 20 .mu.M to about 2 mM. A
surgical rinse of the present invention may be applied before,
during, or after surgery and may be aspirated from the surgical
area or left on the surgical area to inhibit biofilm formation. In
another embodiment, the bandage is impregnated with a chelating
polymer composition.
[0094] In another embodiment of the invention, a method is provided
for inhibiting formation of a biofilm comprising bacteria, the
method comprising contacting the bacteria with an effective amount
of at least one zinc chelator, wherein the bacteria are selected
from the group consisting of Staphylococcus aureus, methicillin
resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis,
methicillin resistant Staphylococcus epidermidis (MRSE),
Streptococcus sanguinis, and Streptococcus suis, whereby formation
of the biofilm is inhibited.
[0095] In still another embodiment of the invention, a method is
provided for inhibiting the formation of a biofilm comprising
bacteria wherein the bacteria comprise at least one zinc adhesion
module, the method comprising contacting the bacteria with a
composition comprising at least one soluble zinc adhesion module,
wherein formation of the biofilm is inhibited. The composition may
be, for example, a spray, lotion, solution, gel, cream, ointment,
surgical rinse, or dental rinse. In another embodiment, the
composition comprising at least one soluble zinc adhesion module
may be a device-soaking solution, a personal cleaning composition,
or a hard surface cleaning composition prepared with recombinant,
purified soluble zinc adhesion modules. In one embodiment, the
composition comprises about 5 .mu.M to about 50 .mu.M of a protein
construct comprising one or two zinc adhesion modules, or about 10
nM to about 10 .mu.M of a protein construct containing three to 17
zinc adhesion modules.
[0096] In another embodiment of the invention, a bandage
impregnated with a safe and effective amount of at least one zinc
chelator is provided, wherein the bandage inhibits the formation of
a biofilm on the skin, wherein the biofilm comprises bacteria
comprising at least one zinc adhesion module. In one embodiment,
the bandage is suitable for use in patients with cuts, burns, turf
burns, abrasions, lacerations, puncture wounds, regions of
bacterial infection such as boils and pustules, and the like. In
another embodiment, the chelator is DTPA and the concentration of
DTPA in the solution impregnating the bandage is from about 20
.mu.M to about 2 mM.
[0097] In another embodiment of the invention, a personal cleansing
composition comprising an effective amount of at least one zinc
chelator is provided, wherein the personal cleansing composition
inhibits formation of a biofilm on the skin, wherein the biofilm
comprises bacteria comprising at least one zinc adhesion module.
Suitable personal cleansing compositions include, but are not
limited to, surgical scrubs, shower gels, body washes, soaps, and
the like. In one embodiment, the chelator is DTPA and the
concentration of DTPA in the personal cleansing composition is from
about 20 .mu.M to about 2 mM. In another embodiment of the
invention, the personal cleansing composition is applied as a part
of a personal hygiene routine. Personal cleansing compositions of
the present invention are suitable for use by a variety of
individuals, including, for example, people recovering from MRSA or
MRSE infections, athletes using team locker rooms, and healthcare
professionals.
[0098] In another embodiment of the invention, a hard surface
cleaning composition comprising an effective amount of at least one
zinc chelator is provided, wherein the composition inhibits
formation of a biofilm on a hard surface, wherein the biofilm
comprises bacteria comprising at least one zinc adhesion module.
The present hard surface cleaning composition has a variety of
useful applications, including use in industrial applications as
well as medical, veterinary, or livestock environments. For
example, hard surface cleaners of the present invention are useful
in the cleaning and treating of pipeline systems, cooling water
systems in power plants, refineries, chemical plants, air
conditioning systems, storage tanks, trays, containers, walls,
floors, countertops, locker room floors, benches, lockers, showers,
bathrooms, toilets, water filtration units, and the like, as part
of a standard cleaning routine. In a specific embodiment, the
chelator is DTPA and the concentration of chelator in the
composition is from about 20 .mu.M to about 20 mM. Optionally, the
at least one zinc chelator is present in the composition in
combination with one or more other cleaning agents, such as
detergents, surfactants, alcohols, hydrochloric or muriatic acid,
acetic acid, sodium or potassium hydroxide, ammonia, and the
like.
[0099] In another embodiment of the invention, a dental rinse is
provided for inhibiting formation of a biofilm wherein the biofilm
comprises bacteria comprising at least one zinc adhesion module,
the dental rinse comprising an effective amount of at least one
zinc chelator.
EXAMPLES
[0100] The following examples are given by way of illustration only
and are not intended to limit the scope of the present
invention.
Example 1
Preparation of DTPA Zinc Chelator Stock Solutions
[0101] A composition according to the present invention comprising
aqueous DTPA in the amounts indicated was prepared:
[0102] A 100 mM stock solution was prepared by dissolving solid
DTPA in 500 mM HCl and incubating at room temperature with
agitation (rocking) for 2 hrs until all powder dissolved. This
concentrated stock solution was used to prepare 8-10.times. stocks
to use for the assay, by diluting concentrated DTPA into filtered
water to give final concentrations 200, 400, 800, 1600, 3200, 6400,
13000, 26000 .mu.M. All chelator solutions were filter-sterilized
by a 0.2 .mu.m nitrocellulose filter prior to use in the biofilm
assay.
Example 2
Preparation of EDTA Zinc Chelator Stock Solutions
[0103] A composition according to the present invention comprising
aqueous EDTA in the amounts indicated was prepared:
[0104] A concentrated 0.5 M stock solution was prepared by
dissolving solid EDTA in filtered water and titrating in 10 M NaOH
dropwise until a pH 8.8 was reached. This concentrated stock
solution was used to prepare 8-10.times. stocks for the assay by
diluting into filtered water and filter sterilizing, as described
in Example 1.
Example 3
Preparation of EDDA Zinc Chelator Stock Solutions
[0105] A composition according to the present invention comprising
aqueous EDDA in the amounts indicated was prepared:
[0106] A concentrated 100 mM stock was prepared by dissolving solid
EDDA in 1N NaOH and titrating in 12 M HCl dropwise until a pH of
8.8 was reached. This concentrated stock solution was used to
prepare 8-10.times. stocks for assay by diluting into filtered
water and filter sterilizing, as described in Example 1.
Example 4
Preparation of DEDTC Zinc Chelator Stock Solutions
[0107] A composition according to the present invention comprising
aqueous DEDTC in the amounts indicated was prepared:
[0108] A concentrated 100 mM stock was prepared by dissolving solid
DEDTC in filtered water. This concentrated stock solution was used
to prepare 8-10.times. stocks for assay by diluting into filtered
water and filter sterilizing, as described in Example 1.
Example 5
Preparation of TPEN Zinc Chelator Stock Solutions
[0109] A composition according to the present invention comprising
aqueous TPEN in the amounts indicated was prepared:
[0110] A concentrated 100 mM stock was prepared by dissolving solid
TPEN in 100% ethanol. This concentrated stock solution was used to
prepare 8-10.times. stocks for assay by diluting into 100% ethanol
and filter sterilizing, as described in Example 1.
Example 6
Preparation of 1,10-Phenanthroline Zinc Chelator Stock
Solutions
[0111] A composition according to the present invention comprising
aqueous 1,10-phenanthroline in the amounts indicated was
prepared:
[0112] A concentrated 100 mM stock was prepared by dissolving solid
1,10-phenanthroline in 100 mM HCl. This concentrated stock solution
was used to prepare 8-10.times. stocks for assay by diluting into
filtered water and filter sterilizing, as described in Example
1.
Example 7
Preparation of Crystal Violet Stock Solution
[0113] A composition according to the present invention comprising
aqueous crystal violet in the amounts indicated was prepared:
[0114] A stock solution was prepared by dissolving 1 gram of solid
crystal violet in 1 liter of filtered water, and passing the
solution through a 0.45 .mu.m nitrocellulose filter.
Example 8
Effectiveness of Zinc Chelators Against Biofilm Formation by
Methicillin-Resistant Staphylococcus epidermidis Strain RP62a
[0115] S. epidermidis strain RP62a (ATCC 35984) was obtained as a
glycerol stock from ATCC. 3 ml overnight cultures were grown in
tryptic soy broth (TSB, BD Biomedical), shaking at 37.degree. C.,
and cultures were diluted 1:200 in TSB for use in the biofilm
assay.
[0116] Each chelator (prepared in Examples 1-6) was added directly
to a 96 well microtiter plate in a series of dilutions. 20 .mu.l of
each stock solution (200, 400, 800, 1600, 3200, 6400, 13000, or
26000 .mu.M chelator) was added to each well. 200 .mu.l of the
1:200 dilution of RP62a bacterial culture (as previously specified)
was added to each well. Controls with bacterial culture media only
and bacterial culture media plus vehicle controls only (i.e., final
concentrations 10 mM HCl, 10% ethanol, or 10 mM NaOH) were added to
wells alone without chelator. The 96 well plates were then
incubated 18-24 hours statically at 37.degree. C. Media was
aspirated by pipet and wells were washed twice with filtered water.
The plates were dried by inversion for 1 hour, and stained with
0.1% crystal violet for 15 minutes. Crystal violet was then
aspirated by pipet and washed once with filtered water. The plates
were then read on a spectrophotometer at a wavelength of 570 nm. An
absorbance greater than 0.1 indicates biofilm formation.
[0117] For RP62a, biofilms were diminished but not cleared by 20
.mu.M DTPA, 40 .mu.M EDTA, 80 .mu.M 1,10-phenanthroline, 20 .mu.M
DEDTC, and 80 .mu.M EDDA. RP62a biofilms were inhibited (i.e.,
absorbance of 0.1 or less) by 40 .mu.M DTPA, 20 .mu.M TPEN, 320
.mu.M EDTA, 640 .mu.M 1,10-phenanthroline, and 1.3 mM DEDTC.
Example 9
Effectiveness of Zinc Chelators Against Biofilm Formation by
Staphylococcus aureus Strain Rosenbach SA113
[0118] S. aureus strain Rosenbach SA113 (ATCC 35556) was obtained
as a glycerol stock from ATCC. 3 ml overnight cultures were grown
in blood-heart infusion broth (BHI, BD biomedical) shaking at
37.degree. C., where cultures were diluted 1:50 for use in the
biofilm assay.
[0119] 2% glucose (20 .mu.l from a 20% glucose stock) was added to
each well prior to the addition of bacterial culture to promote
biofilm formation. In addition, 96 well microtiter plates were
pre-treated with fetal bovine serum (FBS) at 4.degree. C. for at
least 24 hours prior to use. The FBS was aspirated prior to
addition of chelators and glucose for the assay. Each chelator
(prepared in Examples 1-6) was added directly to a 96 well
microtiter plate in a series of dilutions. 20 .mu.l of each stock
solution (200, 400, 800, 1600, 3200, 6400, 13000, or 26000 .mu.M
chelator) was added to each well. 200 .mu.l of the 1:50 dilution of
SA113 bacterial culture (as previously specified) was added to each
well. Controls with bacterial culture media only and bacterial
culture media plus vehicle controls only (i.e., final
concentrations 10 mM HCl, 10% ethanol, or 10 mM NaOH) were added to
wells alone without chelator. The 96 well plates were then
incubated 36-40 hours statically at 37.degree. C. Media was
aspirated by pipet and wells were washed twice with filtered water.
The plates were dried by inversion for 1 hour, and stained with
0.1% crystal violet for 15 minutes. Crystal violet was then
aspirated by pipet and washed once with filtered water. The plates
were then read on a spectrophotometer at a wavelength of 570 nm. An
absorbance greater than 0.1 indicates biofilm formation.
[0120] For SA113, biofilms were diminished but not cleared by 20
.mu.M TPEN, 320 .mu.M EDTA, 320 .mu.M 1,10-phenanthroline, 80 .mu.M
DEDTC, and 20 .mu.M EDDA. SA113 biofilms were inhibited (i.e.,
absorbance of 0.1 or less) by 20 .mu.M DTPA, 40 .mu.M TPEN, 1.3 mM
1,10-phenanthroline, 80 .mu.M DEDTC, and 640 .mu.M EDDA.
Example 10
Effectiveness of Zinc Chelators Against Biofilm Formation by
Staphylococcus aureus Strain COL
[0121] S. aureus strain COL was obtained directly from Brian
Wilkinson (Illinois State University) who has maintained the
culture as a frozen stock since 1976. 3 ml overnight cultures were
grown in blood-heart infusion broth (BHI) shaking at 37.degree. C.,
where cultures were diluted 1:25 for use in the biofilm assay.
[0122] 2% sucrose (20 .mu.l from a 20% sucrose stock) was added to
each well prior to the addition of the bacterial culture to promote
biofilm formation. In addition, 96 well microtiter plates were
pre-treated with fetal bovine serum (FBS) at 4.degree. C. for at
least 24 hours prior to use. The FBS was aspirated prior to
addition of chelators and sucrose for the assay. Each chelator
(prepared in Examples 1-6) was added directly to a 96 well
microtiter plate in a series of dilutions. 20 .mu.l of each stock
solution (200, 400, 800, 1600, 3200, 6400, 13000, or 26000 .mu.M
chelator) was added to each well. 200 .mu.l of the 1:25 dilution of
COL bacterial culture (as previously specified) was added to each
well. Controls with bacterial culture media only and bacterial
culture media plus vehicle controls only (i.e, final concentrations
10 mM HCl, 10% ethanol, and 10 mM NaOH) were added to wells alone
without chelator. The 96 well plates were then incubated 36-40
hours statically at 37.degree. C. Media was aspirated by pipet and
wells were washed twice with filtered water. The plates were dried
by inversion for 1 hour, and stained with 0.1% crystal violet for
15 minutes. Crystal violet was then aspirated by pipet and washed
once with filtered water. The plates were then read on a
spectrophotometer at a wavelength of 570 nm. An absorbance greater
than 0.1 indicates biofilm formation.
[0123] For COL, biofilms were diminished but not cleared by 20
.mu.M EDTA, 80 .mu.M 1,10-phenanthroline, 20 .mu.M DEDTC, and 160
.mu.M EDDA. COL biofilms were inhibited (i.e., absorbance of 0.1 or
less) by concentrations greater than 20 .mu.M DTPA, 20 .mu.M TPEN,
160 .mu.M EDTA, 320 .mu.M 1,10-phenanthroline, and 80 .mu.M DEDTC.
20.
Example 11
Effectiveness of Zinc Chelators Against Biofilm Formation by
Methicillin-Resistant Staphylococcus aureus Strain USA300
[0124] S. aureus strain USA300 was obtained from Daniel Hassett
(University of Cincinnati) as a glycerol stock. 3 ml overnight
cultures were grown in blood-heart infusion broth (BHI) shaking at
37.degree. C., where cultures were diluted 1:25 for use in the
biofilm assay. 2% glucose (20 ul from a 20% glucose stock) was
added to each well prior to the addition of bacterial culture to
promote biofilm formation. In addition, 96 well microtiter plates
were pre-treated with human fibronectin (BD biomedical purchased
from Fisher scientific). The fibronectin was aspirated prior to
addition of chelators and glucose for the assay. Each chelator
(prepared in Examples 1-6) was added directly to a 96 well
microtiter plate in a series of dilutions. 20 .mu.l of each stock
solution (200, 400, 800, 1600, 3200, 6400, 13000, or 26000 .mu.M
chelator) was added to each well. 200 .mu.l of the 1:25 dilution of
USA300 bacterial culture dilution (as previously specified) were
added to each well. Controls with bacterial culture media only and
bacterial culture media plus vehicle controls only (final
concentrations 10 mM HCl, 10% ethanol, and 10 mM NaOH) were added
to wells alone without chelator. The 96 well plates were then
incubated 36-40 hours statically at 37.degree. C. Media was
aspirated by pipet and wells were washed twice with filtered water.
The plates were dried by inversion for 1 hour, and stained with
0.1% crystal violet for 15 minutes. Crystal violet was then
aspirated by pipet and washed once with filtered water. The plates
were then read on a spectrophotometer at a wavelength of 570 nm. An
absorbance greater than 0.1 indicates biofilm formation.
[0125] For USA300, biofilms were diminished but not cleared by 20
.mu.M DTPA, 20 .mu.M TPEN, 1.3 mM EDTA, and 160 .mu.M
1,10-phenanthroline. USA300 biofilms were inhibited (i.e.,
absorbance of 0.1 or less) by concentrations greater than 20 .mu.M
DTPA, 20 .mu.M TPEN, 2.6 mM EDTA, and 1.3 mM
1,10-phenanthroline.
Example 12
[0126] A prosthetic knee replacement is prepared prior to surgery
by bathing the device for 60 minutes in a 2 mM sterile DTPA
solution. The solution is an aqueous solution containing 10 mM HCl,
a vehicle used to solubilized DTPA. When the surgeon is ready to
insert the prosthetic knee replacement, the device is removed from
the sterile DTPA solution and implanted into the patient. Treatment
with the DTPA solution inhibits biofilm formation on the prosthetic
device.
Example 13
[0127] During the surgical installation of an implantable medical
device, the vicinity of the surgical operation is rinsed with a
sterile buffered saline solution containing 100 .mu.M DTPA. Upon
the conclusion of the surgery, the sterile solution is aspirated
from the surgical site. Rinsing with the DTPA solution inhibits
biofilm formation on the implantable device by commensal or
environmental bacteria that gain access to the implantation site
during surgery.
Example 14
[0128] A topical pharmaceutical cream containing 100 .mu.M DTPA is
prepared. This composition is applied to the surface of the skin
after a turf burn injury. Treatment with the topical pharmaceutical
cream inhibits biofilm formation upon infection of the injured area
with methicillin resistant Staphylococcus aureus.
Example 15
[0129] A bandage is impregnated with a pharmaceutical gel
containing 100 .mu.M DTPA. The bandage is applied to the surface of
the skin over a laceration. Treatment with the bandage inhibits
biofilm formation upon infection of the injured area with
Staphylococcus epidermidis.
Example 16
[0130] A personal cleaning composition in the form of a shower gel
containing 1 mM DTPA is prepared. The personal cleaning composition
is applied as a part of a personal hygiene routine by athletes in
the locker room. The DTPA-containing shower gel prevents
biofilm-related infections such as MRSA or MRSE.
Example 17
[0131] A hard surface cleaning composition is prepared containing 2
mM DTPA The cleaning solution is used to soak hard objects such as
surgical instruments or food preparation tools. Biofilm formation
by bacteria having zinc adhesion modules is inhibited.
Example 18
[0132] A hard surface cleaning composition is prepared containing 2
mM DTPA The cleaning solution is applied to pipeline systems.
Application of the cleaning solution prevents the formation of
environmental or industrial biofilms on the surface of the
pipes.
Example 19
[0133] A prosthetic hip replacement is prepared prior to surgery by
bathing the device for 60 minutes in a sterile solution containing
a 25 .mu.M concentration of a protein construct comprising one zinc
adhesion module. When the surgeon is ready to insert the prosthetic
hip replacement, the device is removed from the sterile zinc
adhesion module solution and implanted into the patient. Treatment
with the zinc adhesion module solution inhibits biofilm formation
on the prosthetic device.
Example 20
[0134] During the surgical installation of an implantable medical
device, the vicinity of the surgical operation is rinsed with a
sterile buffered saline solution containing a 2 .mu.M concentration
of a protein construct comprising six zinc adhesion modules. Upon
the conclusion of the surgery, the sterile solution is aspirated
from the surgical site. Rinsing with the zinc adhesion module
solution inhibits biofilm formation on the implantable device by
commensal or environmental bacteria that gain access to the
implantation site during surgery.
[0135] While particular embodiments of the present invention have
been illustrated and described, it would be obvious to one skilled
in the art that various other changes and modifications can be made
without departing from the spirit and scope of the invention. It is
therefore intended to cover in the appended claims all such changes
and modifications that are within the scope of this invention.
Sequence CWU 1
1
21208PRTStaphylococcus epidermidisMISC_FEATURE(1)..(208)Single
complete zinc adhesion module including C-terminal capping sequence
from the Accumulation-associated protein (Aap). 1Val Asp Gly Asp
Ser Ile Thr Ser Thr Glu Glu Ile Pro Phe Asp Lys1 5 10 15Lys Arg Glu
Phe Asp Pro Asn Leu Ala Pro Gly Thr Glu Lys Val Val 20 25 30Gln Lys
Gly Glu Pro Gly Thr Lys Thr Ile Thr Thr Pro Thr Thr Lys 35 40 45Asn
Pro Leu Thr Gly Glu Lys Val Gly Glu Gly Glu Pro Thr Glu Lys 50 55
60Ile Thr Lys Gln Pro Val Asp Glu Ile Val His Tyr Gly Gly Glu Gln65
70 75 80Ile Pro Gln Gly His Lys Asp Glu Phe Asp Pro Asn Ala Pro Val
Asp 85 90 95Ser Lys Thr Glu Val Pro Gly Lys Pro Gly Val Lys Asn Pro
Asp Thr 100 105 110Gly Glu Val Val Thr Pro Pro Val Asp Asp Val Thr
Lys Tyr Gly Pro 115 120 125Val Asp Gly Asp Ser Ile Thr Ser Thr Glu
Glu Ile Pro Phe Asp Lys 130 135 140Lys Arg Glu Phe Asp Pro Asn Leu
Ala Pro Gly Thr Glu Lys Val Val145 150 155 160Gln Lys Gly Glu Pro
Gly Thr Lys Thr Ile Thr Thr Pro Thr Thr Lys 165 170 175Asn Pro Leu
Thr Gly Glu Lys Val Gly Glu Gly Lys Ser Thr Glu Lys 180 185 190Val
Thr Lys Gln Pro Val Asp Glu Ile Val Glu Tyr Gly Pro Thr Lys 195 200
2052208PRTStaphylococcus aureusMISC_FEATURE(1)..(208)Single
complete zinc adhesion module including C-terminal capping sequence
from the SasG protein. 2Val Lys Gly Asp Ser Ile Val Glu Lys Glu Glu
Ile Pro Phe Glu Lys1 5 10 15Glu Arg Lys Phe Asn Pro Asp Leu Ala Pro
Gly Thr Glu Lys Val Thr 20 25 30Arg Glu Gly Gln Lys Gly Glu Lys Thr
Ile Thr Thr Pro Thr Leu Lys 35 40 45Asn Pro Leu Thr Gly Glu Ile Ile
Ser Lys Gly Glu Ser Lys Glu Glu 50 55 60Ile Thr Lys Asp Pro Val Asn
Glu Leu Thr Glu Phe Gly Gly Glu Lys65 70 75 80Ile Pro Gln Gly His
Lys Asp Ile Phe Asp Pro Asn Leu Pro Thr Asp 85 90 95Gln Thr Glu Lys
Val Pro Gly Lys Pro Gly Ile Lys Asn Pro Asp Thr 100 105 110Gly Lys
Val Ile Glu Glu Pro Val Asp Asp Val Ile Lys His Gly Pro 115 120
125Lys Thr Gly Thr Pro Glu Thr Lys Thr Val Glu Ile Pro Phe Glu Thr
130 135 140Lys Arg Glu Phe Asn Pro Lys Leu Gln Pro Gly Glu Glu Arg
Val Lys145 150 155 160Gln Glu Gly Gln Pro Gly Ser Lys Thr Ile Thr
Thr Pro Ile Thr Val 165 170 175Asn Pro Leu Thr Gly Glu Lys Val Gly
Glu Gly Gln Pro Thr Glu Glu 180 185 190Ile Thr Lys Gln Pro Val Asp
Lys Ile Val Glu Phe Gly Gly Glu Lys 195 200 205
* * * * *